TW201110977A - Compositions and methods for preparing staphylococcus aureus serotype 5 and 8 capsular polysaccharide conjugate immunogenic compositions - Google Patents

Abstract

The present invention relates to immunogenic conjugates comprising S. aureus serotype 5 and 8 capsular polysaccharides conjugated to carrier proteins and methods for their preparation and use. Methods for making the immunogenic conjugates of the invention involve covalent conjugation of the capsular polysaccharides with the carrier proteins using conjugation chemistry involving either 1, 1-carboyl-di-1, 2, 4-triazole (CDT) or 3-(2-pyridyldithio)-propionyl hydrazide (PDPH).

Description

201110977 VI. INSTRUCTIONS OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention generally relates to S. aureus (plus flMrews) serotype 5 and 8 capsular polysaccharide conjugate immunogenic compositions and methods of making and using same. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to US Provisional Patent Application Nos. 61/219,143, and No. 61/219,151, the entire disclosure of each of which is incorporated by reference in its entirety. The manner is incorporated herein. [Prior Art] Humans are natural reservoirs of Gram-positive Staphylococcus aureus. For example, S. aureus can be permanently or temporarily colonized in the skin, nostrils, and throat without causing disease. Staphylococcus aureus infections range from mild skin infections to endocarditis, osteomyelitis, bacteremia and septicaemia. Staphylococcus aureus also causes most nosocomial infections and is increasingly prevalent in community-initiated infections. In addition, in 2005, the number of people infected with bis- phthalicillin-resistant Staphylococcus aureus (MRSA) was estimated to be 31.8 per 100,000 individuals, including 16,650 deaths in the United States in 2005 (KIevens et al., (2007) J• version from the heart Qing 298: 1763 1771). When an individual becomes immunocompromised due to destruction of the immune barrier, such as during surgery, placement of an indwelling catheter or other device, trauma or trauma, the disease occurs. Staphylococcus aureus produces a large number of extracellular antigens and intracellular antigens including: polytoxins and enzymes. Of particular interest to this article is the S. aureus capsular sero-serotype (see Karakawa and Vann, "Capsular 149159.doc 201110977 polysaccharides of Siap/^/ococcw Weinstein and

Edited by Fields. Seminars in Infectious Disease. IV. Bacterial Vaccines. (New York, NY; Thieme Stratton; 1982. pp. 285-293), especially serotype 5 and 8 capsular polysaccharides. Epidemiological studies of a large number of strains of Staphylococcus aureus isolates isolated from individuals have shown that between 7〇 and 8〇% are serotype 5 or 8 capsular polysaccharides (Arbeit et al., (1984) / «/eci. Db. 2:85-91). Unfortunately, the capsular polysaccharide itself is a poor immunogen. With the use of intravascular devices and invasive procedures, staphylococcal infections and diseases have increased significantly in the last 20 years. Increasing incidence is more troublesome due to the parallel increase in antibiotic resistance, and therefore there is an urgent need for an immunogenic composition that can prevent staphylococcal infection and disease. SUMMARY OF THE INVENTION The present invention relates to immunogenic conjugates comprising S. aureus serotype 5 or 8 capsular polysaccharides in combination with a carrier protein, and methods of making such conjugates. S. aureus serotype 5 or 8 capsular polysaccharide can be obtained directly from the bacterium using a separation procedure known to those skilled in the art, can be produced using synthetic protocols, or can be genetically engineered as is known to those skilled in the art. The program is reorganized. Further, the present invention provides a method of inducing an immune response against Staphylococcus, a method of preventing a disease caused by Staphylococcus, and a method of reducing the severity of at least one symptom of a disease caused by Staphylococcus infection. In one embodiment The present invention comprises an immunogenic polysaccharide-protein conjugate comprising a combination of a S. aureus serotype 5 or 8 capsular polysaccharide and a 149159.doc 201110977 carrier protein, wherein the molecular weight of the polysaccharide is 2 〇 〇 & l〇〇〇kDa. In some embodiments, the immunogenic conjugate has a molecular weight of from 5 k kaa. In one embodiment, the polysaccharide moiety of the immunogenic conjugate has a molecular weight ranging from 70 kDa to 300 kDa. In one embodiment, the immunogenic conjugate has a molecular weight ranging from 5 〇〇 kDa to 25 〇〇 kDa. In one embodiment, the serotype 5 or 8 capsular polysaccharide has a hydrazine degree of from 10% to 100%. In one embodiment, the degree of 〇_乙醯 is from 5〇% to 100%. In one embodiment, the degree of enthalpy is from 75% to 1%. In one embodiment, the immunogenic conjugate produces a functional antibody, as measured in an animal potency model or via an opsonophagotictic killing assay, based on killing the bacteria. In one embodiment, the immunogenic conjugate vector protein comprises CRM, 97. In one embodiment, CRM97 is covalently attached to the polysaccharide via an amine phthalate linkage, a guanamine linkage, or both. In one embodiment, the molar ratio of the combined lysine to CRM 197 can range from about 1 〇:1 to about 2:5:1. In one embodiment, the conjugate comprises a covalent bond between CRIV^97 and the polysaccharide for at least every 5 to 10 sugar repeating units of the polysaccharide. In one embodiment, the bond between the carrier protein and the polysaccharide occurs once every 5 repeating units of the polysaccharide. In one embodiment, the immunogenic conjugate comprising CRM 97 comprises 5 to 22 lysines or 8 to 15 lysines covalently attached to the polysaccharide. In one embodiment, the immunogenic conjugate comprising CRM!97 comprises 5 to 23 isophthalic acids or 8 to 12 isoleucine covalently linked to the polysaccharide. 149159.doc 201110977 In one embodiment, the immunogenic conjugate comprises 10% to 1% 〇 醯 醯 5 5 or type 8 polysaccharide. In one embodiment, the immunogenic conjugate comprises 3 50 /〇 to 100% 〇_乙醯化5 or 8 type polysaccharide. In one embodiment, the immunogenic conjugate comprises from 75% to 1% hydrazine-type 5 or 8 polysaccharide. In some embodiments, the immunogenic composition can be used to produce a functional antibody in an animal efficacy model or a phagocytic killing assay. In one embodiment, the immunogenic composition comprises less than about 30% free 5 or 8 polysaccharide, based on the total amount of the 5 or 8 polysaccharide. In one embodiment, the immunogenic composition comprises less than about 20% free 5 or 8 polysaccharide, based on the total amount of the 5 or 8 polysaccharide. In one embodiment, the invention comprises an immunogenic composition, such as an immunogenic conjugate as described herein, and at least one adjuvant, diluent or carrier. The adjuvant may be a chain-based adjuvant such as, for example, selenate, sulphate, and hydroxide. In one embodiment, the adjuvant comprises a phytic acid. In one embodiment, the immunogenic composition comprises less than about 30% free 5 or 8 polysaccharide, based on the total amount of the 5 or 8 type polysaccharide. In one embodiment, the immunogenic composition comprises less than about 20% free 5 or 8 polysaccharide, based on the total amount of the 5 or 8 type polysaccharide. In one embodiment, the invention comprises a method of inducing an individual to produce an immune response against a S. aureus serotype 5 or 8 smear multi- vine conjugate, the method comprising administering to the individual an immunologically effective amount as herein Said immunogenic composition. In one embodiment, the invention comprises a method of producing an immunogenic polysaccharide-149159.doc 201110977 protein conjugate comprising separating a S. aureus serotype 5 or 8 capsular polysaccharide from a carrier protein, The method comprises the steps of: reacting isolated S. aureus serotype 5 or 8 capsular polysaccharide with U-mono-di-(1,2,4-triazole) (CDT) in an organic solvent to produce activated serum Type 5 or 8 polysaccharide; and reacting the activated serotype 5 or 8 polysaccharide with the carrier protein in an organic solvent to produce a serotype 5 or 8 polysaccharide: carrier protein conjugate. In one embodiment, the method of activating S. aureus serotype 5 or 8 capsular polysaccharide further comprises lyophilizing the serotype 5 or 8 polysaccharide and resuspending the lyophilized polysaccharide in an organic solvent. In one embodiment, the resuspended polysaccharide is activated and then directly reacted with the carrier protein. In one embodiment, the activated isolated serotype 5 or 8 polysaccharide is isolated and reacted with a carrier protein. In one embodiment, the isolated isolated serotype 5 or 8 sugar is separated by lyophilization to produce a lyophilized activated serotype 5 or 8 polysaccharide, which is then reacted with the carrier protein. In one embodiment, the separation is generated^

The step of resuspending the dried carrier protein in an organic solvent as a separation of the activated serotype 5 or 8 polysaccharide fish alley. New 〇

149159.doc The halo 5 or 8 capsular activated polysaccharide is maintained at a pH of at least about 4 hours at about 8.8 to 201110977 for about 9.2. In one embodiment, the reaction mixture of activated S. aureus ... type labyrinth and carrier protein is maintained at about hunger for about at least 4 hours at about yaw. In one embodiment, the method of producing a S. aureus isolate, such as a type of smear membrane, comprises the step of isolating the cleavage of the A-form protein conjugate after it has been produced. In one embodiment, the organic solvent used in the method for producing a S. aureus (4) type capsular polysaccharide: carrier protein conjugate is a very non-physiological bath. In one embodiment, the polar aprotic solvent is selected from the group consisting of monomethyl hydrazine (DMSO). In one embodiment, the organic solvent in the method of producing a sugar-carrier protein conjugate is DMS0. In a uniform embodiment, a method of producing a S. aureus Type 5 capsular multi-carrier protein conjugate comprises adjusting a water concentration of a reaction mixture comprising a Type 5 capsular polysaccharide and an organic solvent to about 〇1% As for the steps of .3 /〇. In one embodiment, the water concentration of the reaction mixture comprising the Type 5 capsular polysaccharide and cDT in an organic solvent is adjusted to about 0.2%/0. In one embodiment, the step of activating the isolated S. aureus Type 5 capsular polysaccharide comprises reacting the polysaccharide with Cdt, wherein the CE)T is used in an amount greater than the reaction mixture of the 匕35 capsular polysaccharide and the CDT in an organic solvent. The amount of polysaccharide present in the excess is 20 moles. In one embodiment, the method of producing a S. aureus Type 8 capsular occlusion protein binding comprises the step of determining the water concentration of a reaction mixture comprising Type 8 capsular polysaccharide 149159.doc 201110977. In one embodiment, the amount of CDT added to the reaction mixture to activate the polysaccharide is provided in accordance with the amount of CDT equivalent to about the molar amount of water in the reaction mixture comprising the type 8 serotonin and the CDT in an organic solvent. In one embodiment, the amount of CDT added to the reaction mixture to activate the polysaccharide is about 5:1 according to the molar ratio of water in the reaction mixture comprising the type 8 capsular polysaccharide and CDT in an organic solvent. The amount of CDT is provided. In one embodiment, the amount of CDT added to the reaction mixture to activate the polysaccharide is in accordance with a cdt having a water content of about 751 in a reaction mixture comprising a type 8 capsular polysaccharide and a CDT in an organic solvent. Quantity provided. In one embodiment, the method comprising the step of isolating the activated polysaccharide comprises a diafiltration step. In one embodiment, the method comprises lyophilizing the carrier protein, lyophilizing the dialysis carrier protein relative to NaC1 and adjusting the weight ratio (W/W) of the NaCl/carrier protein to about 〇·5 to About 1.5. In one embodiment, the ratio of NaCl to carrier protein is about ι. Capsules In the conjugate example, the carrier protein used in the method for isolating S. aureus 5 or 8 polysaccharide-carrier protein conjugates contains CRMm. In one embodiment, the polysaccharide-carrier protein serotype 5 or 8 polysaccharide reacts in a weight ratio of CRM97 and activated 1:1 used in a method for producing a S. aureus 5 or 8 capsular compound. . In one embodiment, the method of producing a S. aureus 5 or 8 type capsule is disclosed. 149159.doc 201110977 The method of the two-carrier protein conjugate comprises the step of mixing the (4) type of money with the CDT and mixing with the CDT for organic spraying. Icicle sugar. In the case of 'in the case of producing a S. aureus 5 or 8 type membrane two carrier egg two nucleus, the conjugate comprises hydrolyzing a serotype 5 or more conjugates to remove unreacted activity. The steps of the group. In one embodiment, the invention provides a method of producing an immunogenic conjugate comprising separating a S. aureus serum membrane cholestasis binding to a carrier protein, the method comprising the steps of: making = yellow-cocci Serotype 5 or 8-inch membrane and 3H-derived disulfide)-propionate (PDPH) and carbodiimide are reacted in an organic solvent to produce a PDPH-linked multi-audio; the pDpH is linked to a reducing agent and a reducing agent. The reaction produces an activated polysaccharide; the activated serotype 5 or 8 polysaccharide is isolated to produce an isolated activated serotype 5 or 8 polysaccharide; an activated carrier protein is provided; and the isolated activated serotype 5 or 8 polysaccharide is reacted with an activated carrier protein to produce a serotype $ or 8 multi-carrier protein conjugate; thereby producing an immunogenic conjugate comprising a S. aureus serotype 5 or 8 serotonin polysaccharide in combination with a carrier protein f. In one embodiment, after activating the carrier protein, the activated carrier protein is reacted with the activated polysaccharide. In one embodiment, the step of isolating the activated carrier protein further comprises lyophilizing the activated serotype 5 or 8 polysaccharide' to produce a lyophilized activated serotype 5 or 8 polysaccharide. In one embodiment, the bromoacetic acid is N-hydroxybutylimine imide (BAANS) of bromoacetic acid. In one embodiment, the method of producing a serotype 8 capsular polysaccharide using PDPH - 149159.doc -10- 201110977 bulk protein conjugate comprises using an organic solvent which is a polar aprotic solvent. In one embodiment, the polar aprotic solvent is selected from the group consisting of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimercaptoacetamide, methyl-2-pyrrolidone, and six A group consisting of mercaptophosphoramide (HMPA). In one embodiment, the organic solvent is disulfoxide (DMSO). In one embodiment, the carbodiimide used in the method of producing a serotype 5 or 8 capsular polysaccharide-carrier protein conjugate using PDPH is 1-ethyl-3-(3-didecylaminopropyl) ) - carbodiimide (EDAC). In one embodiment, the method of producing a serotype 5 or 8 capsular polysaccharide-carrier protein conjugate using PDPH and EDAC comprises serotype 5 or 8 capsular polysaccharide with PDPH and EDAC at a ratio of about 1:5:3: PDPH: The step of EDAC weight ratio in the reaction in an organic solvent. In one embodiment, the reducing agent used in the method of producing a serotype 5 or 8 capsular polysaccharide-carrier protein conjugate using PDPH and EDAC is dithiothreitol (DTT). In one embodiment, the activating vector protein comprises reacting a carrier protein with bromoacetic acid in a method of producing a serotype 5 or 8 capsular polysaccharide-carrier protein conjugate using PDPH and EDAC. In one embodiment, the step of isolating the activated serotype 5 or 8 polysaccharide in a method of producing a serotype 5 or 8 capsular polysaccharide-carrier protein conjugate using PDPH and EDAC comprises diafiltration. In one embodiment, the method of producing a serotype 5 or 8 capsular polysaccharide-carrier protein conjugate using PDPH and EDAC comprises the step of hydrolyzing a serotype 5 or 8 polysaccharide-carrier protein conjugate to remove unreacted reactive groups . 149159.doc 201110977 In one embodiment, the step of hydrolyzing the crude 5 or 8 polysaccharide-carrier protein conjugate comprises adding cysteamine hydrochloride. In one embodiment, the method of producing a serotype 5 or 8 capsular polysaccharide-carrier protein conjugate using PDPH & EDAC further comprises isolating a S. aureus serotype 5 or 8 capsular polysaccharide in combination with a carrier protein Immunogenic conjugate. In one embodiment, the isolated serotype 5 or 8 polysaccharide carrier protein complex comprises diafiltration. In one embodiment, the carrier protein used in the method of producing a serotype 5 or 8 capsular polysaccharide-carrier protein conjugate using PDPH and EDAC comprises crm197. In one embodiment, the CRM 丨97 line in the method of producing a serotype 5 or 8 capsular polysaccharide _CRM97 conjugate using PDPH & EDAC is added at a weight ratio of about 1:1 CRMm: capsular polysaccharide molecule. In one embodiment, the activated 5 or 8 type capsular polysaccharide used in the method of producing a serotype 5 or 8 capsular-carrier protein conjugate using PDPH and EDAC has a molecular weight of from about 50 kd to about 500 kd. In one embodiment, the immunogenic composition produced by the method of producing a serotype 5 or 8 capsular saccharide carrier protein conjugate using PDPH and EDAC has a molecular weight of from about 400 kd to about 5000 kd. In one embodiment, the invention provides an immunogenic composition comprising a 5 or 8 type capsular polysaccharide-carrier protein conjugate produced by any of the methods described herein. In one embodiment, the invention provides an immunogenic composition, 149159.doc -12 201110977 comprising a 5 or 8 type capsular polysaccharide-carrier protein conjugate produced by any of the methods described herein and at least An adjuvant, diluent or carrier. In one embodiment, the immunogenic composition comprises an aluminum-based adjuvant selected from the group consisting of aluminum phosphate, aluminum sulfate, and aluminum hydroxide. In one embodiment, the immunogenic compositions described herein comprise an adjuvant aluminum phosphate. The immunogenic compositions described herein may comprise less than 30% and less than 2% by weight of free 5 or 8 polysaccharides, based on the total amount of the 5 or 8 type polysaccharide. The immunogenic composition described herein can be stored in water or a low ionic strength neutral pH buffer. In a single embodiment, the invention provides a method of reducing or preventing a staphylococcal infection, a disease or condition associated with a bacterium of the bacterium, the method comprising administering to the individual a therapeutic or prophylactic amount as described herein (IV) of the immunogenic composition. In one embodiment, the infection, disease or condition is selected from the group consisting of invasive Staphylococcus aureus, sepsis, and a carrier. In one embodiment, the invention provides a method of reducing or preventing staphylococcal infection in an individual undergoing surgical private order, the method comprising administering to the individual a prophylactically effective amount of the immunization as described herein The step of the original composition. In one embodiment, the method of the invention comprises replacing CDT with CDI. In one embodiment, the invention provides a 4 conjugate of a S. aureus 5 or 8 capsular polysaccharide having a 50 kDa to 800 kDa molecule and a carrier protein; wherein the polysaccharide is covalently bound to the carrier protein The combined molecular weight is from about 400 kE)a to 5000 kDa. 149159.doc 201110977 In one embodiment, the covalent conjugate of the polysaccharide to the carrier protein comprises a polysaccharide moiety having a molecular weight ranging from 70 kDa to 300 kDa. In one embodiment, the covalent conjugate of the polysaccharide to the carrier protein has a molecular weight ranging from 500 kDa to 2500 kDa. In one embodiment, the carrier protein portion of the covalent conjugate of the polysaccharide to the carrier protein comprises CRM!". In one embodiment, CRMi97 is covalently linked via a urethane linkage, a guanamine linkage, or both. To the polysaccharide. In some embodiments, the molar ratio of the combined lysine to CRMi97 is from about 1 :1 to about 25: 1. In some embodiments, the covalent combination of the polysaccharide with the carrier protein is at least at least At least one covalent bond between the CRMm and the polysaccharide is included every 5 to 1 saccharide repeating unit. In some embodiments, the covalent conjugate of the polysaccharide to the carrier protein is at every 5 sugar repeating units of the polysaccharide Containing at least one bond between CRM97 and the polysaccharide. In some embodiments, the CRMi97 moiety in the covalent conjugate of the polysaccharide with CRM, 97 comprises from 5 to 22 lysine covalently linked to the polysaccharide. In embodiments, the portion of CRMi97 in the covalent conjugate of the polysaccharide with CRMw comprises 5 to 23 lysines covalently linked to the polysaccharide. In some embodiments, the CRM in the covalent conjugate of the polysaccharide with the carrier protein] 9? Part contains 8 to 15 covalent connections The lysine of the polysaccharide is attached. In some embodiments, the CRMm portion of the covalent conjugate of the polysaccharide with the carrier protein comprises from 8 to 2 lysines covalently linked to the polysaccharide. In one embodiment, The invention provides an immunogenic composition comprising a covalent conjugate of a S. aureus 5 or 8 polysaccharide and a carrier protein as described herein and at least one adjuvant, diluent or carrier. 149159.doc -14 · 201110977 In one embodiment, the invention provides an immunogenic composition comprising a covalent conjugate comprising a S. aureus 5 or 8 polysaccharide as described herein and a carrier protein to produce an individual as described herein The method of the immunoreaction is described. In one embodiment, the present invention provides a method of isolating a multi-audio having a molecular weight of from 2 〇 kDa to 1000 kDa. In one embodiment, the present invention provides a capsular polysaccharide by the present invention, An antibody produced by an immunogenic conjugate or an immunogenic composition. [Embodiment] The present invention will be more thoroughly understood in consideration of the [embodiment] of the present invention, and The features, aspects and advantages will become apparent. This [embodiment] is based on the following figures. SUMMARY The present invention relates to the immunogenic binding of the polysaccharide containing the golden yellow grape g serotype (8) in combination with the carrier protein. And novel methods for their preparation and use. The novel features of the immunogenic conjugates of the invention include the molecular weight distribution of the polysaccharide and the resulting conjugate, the ratio of bound lysine in each cRm197 carrier protein, and the covalent attachment to the polysaccharide. The number of amine acids, the number of covalent bonds between the carrier protein and the polysynthesis, the relationship between the repeating units of the multiple enzymes, and the relative amount of free polysaccharides based on the total polysaccharide. As used herein, the term "free polym stomach" A polysaccharide that binds to the carrier protein but (d) is present in the conjugate composition. A method for making the immunogenic conjugate of the present invention comprises covalently binding a (4) polysaccharide to a carrier protein f using a binding chemistry method in combination with a chemical reagent package 149159.doc 15 201110977 including CDI (1,1-carbonyldiimidazole), CDT (1,1-Carbonyl-di-1,2,4-triazole) or PDPH (3-(2-pyridyldithio)-propanoid). CDI is only specific for CP8 integration. The use of CDI/CDT produces a single carbon or zero carbon linker between the capsular polysaccharide and the carrier protein and uses PDPH to create a covalent thioether bond between the capsular polysaccharide and the carrier protein. Other crosslinkers used to bond -SH (thiolated CP) to -NH2 include, but are not limited to: sulfo-LC-SMPT; sulfo-LC-SMPT (6-mercapto-a-(2·0) σ 定 二 二 二 ) ) ) ) ] ] ] sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf sulf Sulfobutylidene sulfite); sulf0-LC-SPDP (6-(3,-[2-pyridyldithio; μpropylamino)hexanoic acid sulfobutanediimide), Can be decomposed by thiol; sulf〇_ SMPB (4-[p-butyleneiminophenyl]butyric acid sulfobutane diimide); sulfo-SIAB ([4-iodoethyl) Sulfophenyl sulphonate; sulfo-EMCS ([Ne-m-butylene imino hexyl decyloxy] sulphate succinimide); EMCA (Ne- Cis-butyl diimide iminohexanoic acid); sulfo-SMCC (4-[7V-methylene-2-imidazolylmethyl]cyclohexane ruthenium sulphate; -MBS (m-cis-butyl diimide phenyl sulfonyl hydroxy sulfosyl dimethyl imidate); sulf 〇 GMBS (N_ [g-m-butyleneimine butyl fluorenyloxy] sulfonate Keidine diimine); ΜΡΑ(Ν-β-m-butylenediminopropionic acid); 2-iminothiolane hydrochloride ' 3-(2- ° ratio. thiodithio) propionic acid butyl imidate ; 3- cis-butyl diimide iminopropyl phthalate - butyl succinimide; 4 - maleic acid sulfonium butyrate - butyl sulfoxide; SMPT (4-butane bismuth) Iminooxycarbonyl-fluorenyl-a-[2-pyridyldithio]nonanthene); lc-SMCC (4-[N-cis-butane I49159.doc 201110977 iminomethyl] ring Hexylcarboxyl_[6_decylaminocaproic acid succinimide vinegar]); KMUA (Nk-m-butyleneimidodecanoic acid); LC-SPDP (6-(3-[2- ° ratio of dimethyldithio]- propylamino) hexamethylene hexanoate); — SMPH (6-[P-m-butyleneimine propylamino] hexanoic acid Imine 'vinegar'; SMPB (4-[p-butyleneiminophenyl)butyric acid butyl imidate acetate); SIAB ([4-iodoethenyl]aminobenzoic acid Dimethyl imidate); EMCS (Ne-m-butylene imino hexyl decyloxy) butadiene imidate); SMCC (4-[7\M bebutenyldiimide) Mercapto]butyl iodide cyclohexanecarboxylate); MBS (m-cis-butylene) Aminophenyl fluorenyl-Nyf butyl succinimide ester; SBAP (3-[bromoethylamino) propionic acid butyl succinimide); BMPS (#-[p-cis-butane Iminopropoxy]butanediimide); AMAS (N-(a-m-butyleneimidoethyloxy)butanediamine); SIA (iodoacetate)醯imino ester); and (4 gastric iodine oxime) amino benzoic acid N-butyl succinimide. These reagents can also be crosslinked using a crosslinking agent for the attachment of _SH to the -OH group. Such crosslinking agents include, but are not limited to, PMPI (N_[p-butadienyldiaminophenyl]isocyanate). The compositions and methods described herein are suitable for a variety of applications. For example, a conjugate can be used to produce a conjugate immunogenic composition to protect the recipient from infection by S. aureus. Alternatively, various combinations can be used to generate antibodies against bacterial capsular polysaccharides, which can then be used in research and clinical laboratory analysis, such as bacterial detection and serotype classification. Such antibodies can also be used to confer passive immunity to an individual. In a differential embodiment φ ^ ^ - beta 肀 所 produced by the antibody against bacterial polysaccharides in the animal efficacy model or conditioning table divided into cell kills 149159.doc - 17- 201110977 analysis is functional. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the methods and materials described herein are preferred. In describing the embodiments and claiming the present invention, certain terms will be used in accordance with the definitions set forth below. As used herein, the singular forms " " " " " " Thus, for example, reference to "the method" includes one or more methods and/or steps of the type described herein and/or will be apparent to those of ordinary skill in the art. As used herein, "about" means within a statistically meaningful range of values, such as the concentration range, time range, molecular weight, temperature, or positivity. The circumference may be within an order of magnitude, typically within 20% of a given value or range, more typically within 1G%, and more typically within 5%. The term “about” means the deviation of the material will depend on the particular system being studied and can be easily understood by the general practitioner. Each integer within the scope is also considered to be an embodiment of the invention. It should be noted that in the present invention, terms such as "including", "containing" and the like may have the meaning given to them in the U.S. Patent Law; for example, it may mean "include" and the like. These terms are meant to include a particular ingredient or group of ingredients without excluding any other ingredients. Terms such as "consisting essentially of" have the meaning given to them in U.S. Patent Law, for example, to allow for the inclusion of other 149159.doc.18.201110977 points or steps that do not detract from the novel or essential characteristics of the present invention, that is, , the other undescribed ingredients or steps of the present invention, and = novel or essential features of the present invention, such as those cited herein or by reference to the prior art components or steps, especially in this document? This technology in the article is a patent that can be patented (for example, new, inconspicuous, and creative compared to the prior art, for example, cited in this article or incorporated by reference.) In the case of the examples incorporated herein, the term "composition" is composed of _.. has the meaning given to it in the U.S. Patent Law; that is, the terms are closed. Therefore, such terms are intended to include a particular ingredient or group of ingredients and exclude all other ingredients. Immunogenic conjugates As described above, the present invention relates to immunogenic conjugates comprising a S. aureus serotype 5 or 8 capsular polysaccharide in combination with a carrier protein. One embodiment of the invention provides an immunogenic conjugate comprising a S. aureus serotype 5 or 8 capsular polysaccharide in combination with a carrier molecule or protein having one or more of the following characteristics: the polysaccharide has a 50 kDa to 7 〇 Molecular weight of 0 kDa; the immunogenic conjugate has a molecular weight of from 500 kDa to 2500 kDa; and the conjugate comprises less than about 3% by weight of free polysaccharide relative to the total polysaccharide. In some embodiments, the polysaccharide has a molecular weight of from 2 〇 kDa to 1 〇〇〇 kDa. In some embodiments, the immunogenic conjugate has a molecular weight of from 200 kDa to 5000 kDa. In other embodiments, the conjugate comprises less than about 25%, about 20%, about 15%, about 1%, or about 5% free polyphonic relative to the total polysaccharide. As used herein, "conjugates" encompasses capsular polysaccharides and carrier proteins which typically have a desired molecular weight range, wherein the capsular polysaccharide binds to the carrier 149159.doc-19·201110977 protein. The conjugate may or may not contain free podopolysaccharide in a certain mash. As used herein, "free capsular is biliary," capsular polysaccharide with capsular polysaccharide: carrier protein conjugates that are not co-compensatory (ie, non-covalent sputum. attached or retained). The terms "free capsular polysaccharide", "free = sugar" and "free sugar" are used interchangeably and are intended to mean the same meaning. Regardless of the nature of the carrier molecule, it can be bound directly or via a linker to the labyrinth. As used herein, "binding" refers to the process by which a bacterial laughing membrane (4) is covalently attached to a carrier molecule. The combination enhances the immunogenicity of the fine membranous polysaccharide. Combinations can be made according to the methods described below or by other methods known in the art. When used in an immunogenic composition, the molecular weight of the golden yellow (tetra) cocide polysaccharide is considered. High molecular weight laughing polysaccharides are capable of inducing certain antibody immune responses due to the high valency of the epitopes present on the surface of the antigen. The separation of "high molecular weight capsular polysaccharides" is contemplated for use in the compositions and methods of the present invention. In the embodiment of the present invention, a high molecular weight serotype 5 or 8 capsular polysaccharide having a molecular weight in the range of 20 kDa to 1000 kDa can be isolated and purified. In the present invention - an embodiment, a high molecular weight serotype 5 or 8 capsular polysaccharide having a molecular weight in the range of 5 〇 kDa to 700 kDa can be isolated and purified. In one embodiment of the invention, a still molecular weight serotype 5 or 8 capsular polysaccharide having a molecular weight in the range of 5 〇 kDa to 300 kDa can be isolated and purified. In a practical example, a high molecular weight serotype 5 or 8 capsular polysaccharide having a molecular weight in the range of 7 〇 kDal 3 〇 () kDa can be isolated and purified. In one embodiment, a high molecular weight serotype 5 or 8 capsular polysaccharide having a molecular weight in the range of 90 kDa to 250 kDa can be isolated and purified. In one embodiment, the molecular weight can be separated and purified at I49159.doc •20·201110977 High molecular weight serotype 5 or 8 capsular polysaccharides ranging from 90 kDa to 150 kDa. In one embodiment, a high molecular weight serotype 5 or 8 capsular polysaccharide having a molecular weight in the range of 9 〇 kDa to 120 kDa can be isolated and purified. In one embodiment, a high molecular weight serotype 5 or 8 capsular polysaccharide having a molecular weight in the range of 80 kDal12() kDa can be isolated and purified. Other ranges of high molecular weight serotype 5 or 8 capsular polysaccharides which can be isolated and purified by the method of the invention include molecular weights from 70 kDa to 100 kDa; molecular weights from 7 〇j^a to 110 kDa; molecular weights from 70 kDa to 120 kDa The molecular weight is 7〇kDa to 130 kDa; the molecular enthalpy is 70 kDa to 140 kDa; the molecular weight is 7〇kDa to 150 kDa; the molecular I is 70 kDa to 160 kDa; the molecular weight is 8〇kDa to 110 kDa; the molecular enthalpy is 80 kDa to 120 kDa; molecular weight 80 kDd 13〇kDa; molecular set at 80 kDa to 140 kDa; molecular weight 80 kDa to 150 kDa; molecule 3: 80 kDa to 160 kDa; molecular weight 9〇kDa to 110 kDa; molecular weight From 90 kDa to 120 kDa; molecular weight from 9〇kDa to 130 kDa; molecular weight from 90 kDa to 140 kDa; molecular weight from 90 kDa to 150 kDa; molecular weight from 90 kDa to 160 kDa; molecular weight from 1〇〇kDa to 120 kDa; The molecular weight ranges from 100 kDa to 130 kDa; the molecular weight ranges from 1 〇〇 kDa to 140 kDa; the molecular weight ranges from 100 kDa to 150 kDa; the molecular weight ranges from 1 〇〇 kDa to 160 kDa; and similar molecular weight ranges. Any integer within any of the above ranges is considered to be an embodiment of the invention. In one embodiment, the molecular weight of the conjugate is from about 5 〇 kDa to about 5000 kDa. In one embodiment, the conjugate has a molecular weight of from about 2 〇〇 kDa to about 5000 kDa. In one embodiment, the immunogenic conjugate has a molecular weight of from about 500 kDa to about 25 〇〇 kDa. In one embodiment, the immunogenic knot 149159.doc 21 201110977 has a molecular weight of from about 500 kDa to about 2500 kDa. In one embodiment, the immunogenic binder has a molecular weight of from about 60 〇 kDa to about 2800 kDa. In one embodiment, the immunogenic conjugate has a molecular weight of from about 7 〇〇 kDa to about 2700 kDa. In one embodiment, the immunogenic binder has a molecular weight of from about 1000 kDa to about 2000 kDa; from about 1800 kDa to about 2500 kDa; from about 11 〇〇 kDa to about 2200 kDa; from about 1900 kDa to about 2700 kDa; kDa to about 2400 kDa; about 1700 kDa to about 2600 kDa; about 1300 kDa to about 2600 kDa; about 1600 kDa to about 3000 kDa. Any integer within any of the above ranges is considered to be an embodiment of the invention. As used herein, "immunogenic" means that an antigen (or antigenic determinant of an antigen), such as a bacterial capsular polysaccharide, or a binding immunogenic composition comprising an antigen, elicits a bodily fluid or cell in a host such as a mammal. The ability to mediate or both" Thus, as used herein, "immunogenic conjugate" or "conjugate" means a bacterial capsular polysaccharide antigen or antigenic determinant (ie, an epitope) that binds to a carrier molecule and is useful for eliciting an immune response. Any immunogenic conjugate. Immunogenic conjugates can be used to sensitize hosts by presenting antigens that bind to MHC molecules on the cell surface. In addition, antigen-specific T cells or antibodies can be produced to further protect the immunized host. Thus, an immunogenic conjugate can protect the host from one or more symptoms associated with bacterial infection, or can protect the host from infection by infection with bacteria associated with the capsular polysaccharide. Immunogenic conjugates can also be used to produce multiple strains or monoclonal antibodies' such antibodies can be used to confer immunity to an individual. Immunogenic conjugates can also be used to produce functional antibodies, such as 149l59.doc • 22· 201110977, measured in animal efficacy models or via conditioned pharyngeal killing assays, based on killing bacteria. An "antibody" is an immunoglobulin molecule capable of specifically binding to a stem such as a carbohydrate, a polynucleotide, a lipid, a polypeptide or the like via at least one antigen recognition site located in the variable region of an immunoglobulin molecule. As used herein, unless the context indicates otherwise, it is intended that the term encompasses not only a multiplicity of strains or a single antibody, but also an engineered antibody (eg, an antibody that has an effector function, stability, and other biological activity changes, Humanized antibodies and/or derived antibodies) and fragments thereof (such as Fab, Fab, 'F(ab')2, Fv), single-stranded (ScFv) and domain antibodies (including shark and camelid antibodies) and antibody-containing portions Fusion proteins, multivalent antibodies, multispecific antibodies (eg, bispecific antibodies, as long as they exhibit the desired biological activity) and antibody fragments as described herein, and any other modified immunoglobulin molecules comprising an antigen recognition site structure. Antibodies include any class of antibodies, such as IgG, IgA or IgM (or a subclass thereof), and the antibodies do not have to belong to any particular class. Depending on the amino acid sequence of the antibody heavy chain constant domain, immunoglobulins can be classified into different classes. There are five main classes of immunoglobulins: "A, IgD, IgE, IgG, and IgM, and many of them can be further divided into subclasses (homotypes)' such as IgG1, IgG2, lgG3, IgG4, igAl in humans. The heavy-chain constant domains of the class immunoglobulin are called α, δ, ε, γ, and μ, respectively. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known. The antibody fragment only contains one part of the intact antibody, wherein Partially preferred retention, when present in the intact antibody, is normally associated with the portion of at least one of the functions of 149159.doc -23- 201110977, preferably most or all of the beta term "antigen" generally means A biomolecule in an animal that stimulates the production of an antibody or a 7-cell response or both, typically a protein, peptide, polysaccharide or conjugate in an immunogenic composition or an immunogenic material, including a composition that is injected or absorbed into an animal. . An immune response can be produced against a complete molecule or a different portion of a molecule, such as an epitope or hapten. The term can be used to refer to an individual molecule of an antigenic molecule or to a homologous or heterologous population of antigenic molecules. The antigen can be recognized by antibodies, tau cell receptors or other elements with specific humoral and/or cellular immunity. "Antigens" also include all relevant antigenic epitopes. The epitope of the designated antigen can be identified using epitope characterization techniques well known in the art. See for example Epit〇pe

Mapping Protocols in Methods in Molecular Biology, Vol. 66 (edited by Glenn E. Morris, 1996) Humana Press, Totowa, N.J. For example, a linear epitope can be determined, for example, by simultaneously synthesizing a large straight shape on a solid support. § A metamorphism corresponds to each part of a protein molecule; and while the peptides are still attached to the support, The peptides react with the antibody. Such techniques are known in the art and are described, for example, in U.S. Patent No. 4,708,871, Geysen et al., (1984) /Voc. iVa". sCz. tASZ 81:3998-4002; Geysen et al., ( 1986) Mo/ec. / Vision (10) 0/. 23: 709-715; each of which is incorporated herein by reference in its entirety herein in its entirety in its entirety. Similarly, conformational epitopes can be identified by, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance to determine the spatial configuration of the amino acid. See, for example, Epitope Mapping Protocols, above. Furthermore, for the purposes of the present invention, "antigen" can also be used to include modifications to the native sequence (additions and substitutions of 149159.doc -24 · 201110977 (essentially generally conservative, but which may be n) Protein, as long as the protein can still trigger an immune response. = such modifications may be intentional, such as induced or borrowed by site-directed mutagenesis, or by genetic engineering methods, or by & . Furthermore, the antigen may be derived, obtained or isolated from a microorganism, such as a bacterium or may be a whole organism. Also included in this definition are oligonucleotides or polynucleic acids that exhibit antigens, such as in nucleic acid immunization applications. Also included are synthetic antigens, such as multiple epitopes, flanking epitopes, and other antigens derived from recombinant or synthetic (Bergmann et al., (1993) 5 by j / w (10) / 23: 2777 2781; Bergmann^A ^ (1996) J. Immunol. 157:3242-3249; Suhrbier (1997) Immunol. Cell Biol. 75:4〇2 408; Gardner et al., (1998) 12th w〇rld Ams c〇nference,

Switzerland, June 28 to July 3, 1998). "Protective" immune response refers to the ability of an immunogenic composition to elicit a humoral or cell-mediated or both-mediated immune response to protect an individual from infection. The protection provided need not be absolute, that is, if there is a statistically significant improvement compared to a control individual population (eg, an infected animal that has not been administered a vaccine or immunogenic composition), then it is not necessary to completely prevent or eradicate the infection. . Protection may be limited to reducing the severity or speed of infection. In general, "protective immune response" will include an increase in the amount of an antibody that is specific for a particular antigen in at least 5% of the individual, including some measureable response of the functional antibody to the various antigens. In certain instances, a "protective immune response" can include a two-fold or four-fold increase in the amount of an antibody specific for a particular antigen 149] 59.doc -25- 201110977, including functional antibodies, in at least 50% of individuals. Some degree of response to various antigens can be measured. In certain embodiments, the 〇ps〇nising antibody is associated with a protective immune response. Thus, the protective immune response can be analyzed by measuring the percent decrease in the number of bacteria in the opsonophagocytic assay, such as those described below. The number of bacteria decreased by at least 鸠, 25%, 5G%, 65%, 75%, 85%, 鸠, 95% or 95% or more. The "immunogenic amount" of a particular combination in a composition is generally based on the combination of the combination. For example, in a dose of 1 〇〇mcg, a serotype 5 or 8 capsular polysaccharide conjugate comprising guanidine free polysaccharide will have about 8 〇 (d) bound polysaccharide and about 20 mcg unbound polysaccharide. The proportion of protein in the conjugate is usually not taken into account when calculating the conjugate dose. The amount of the conjugate can vary depending on the serotype of the staphylococcus. In general, each dose will comprise from 1 to (10) W of biliary, from 0.1 to 10 mgg of polysaccharide, and more particularly 1 JL of 1 mcg of polysaccharide.匕 The term “individual” refers to a mammal, bird, fish, reptile or other animal. The term "individual" also includes humans. The words "individuals" also include keeping pets. Non-limiting examples of domestic pets include: dogs, cats, snails, rabbits, rats, mice, gerbils, stagnation, guinea pigs, shoals, blacks, snakes, gradual fields, fish, turtles, and baby. The term "individual" also includes livestock. Non-limiting examples of livestock include: dried sheep, bison, road dry, cattle, deer, pigs, horses, road horses, scorpions, crickets, sheep, goats, rabbits, deer cattle, chickens, geese and turkeys. As shown in Figure 1, S. aureus serotypes 5 and 8 capsular _ have X lower, · mouth blood / month type 5 [-4] _p_D-ManNAcA-(l-4)-3-〇 , VIII149l59.doc •26- 201110977 α-L-FucNAc-(l-3)-β-DFucNAc-(l->]/^ and serotype 8[-3]-4-0-Ac-PD- ManNAcA-(l-»>3)-aL-FucNAc-(l->-3)-p-DFucNAc-(l-]«. See Jones (2005) Less. Heart 340: 1097-1106. Serum Type 8 capsular polysaccharide has a trisaccharide repeat unit similar to serotype 5 capsular polysaccharide; however, it differs in sugar linkage and 0-acetylation site, thereby producing a serologically different immune response pattern (Fournier Et al., (1984) /wm(10).45:87-93; and Moreau et al. (1990) 201:285-297). Thus, serotype 8 and 5 capsular polysaccharides are relatively complex carbohydrates that are water soluble. Sexual, usually acidic and previously thought to have a molecular weight of about 25 kDa (Fattom (1990) 58, 2367-2374). In some embodiments, the serotype 5 and/or 8 capsular polysaccharide of the invention is subjected to 0-B In some embodiments, the 5-acetylation degree of type 5 capsular polysaccharide or oligosaccharide is from 10% to 100%. %, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 50% to 90%, 60% to 90% '70% to 90% or 80% to 90%. In some embodiments, the 8-type capsular polysaccharide or oligosaccharide has a 0-acetylation degree of 10°/ 〇 to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 50% to 90%, 60% to 90%, 70% to 90% or 80% to 90%. In some embodiments, 0-acetylation of type 5 and type 8 capsular polysaccharides or oligosaccharides Degrees are 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100% , 90% to 100%, 50% to 90%, 60% to 149159.doc -27- 201110977 90%, 70°/. To 90°/. Or 80% to 90%. The 0-acetylation degree of a succulent or oligosaccharide can be determined by any method known in the art, for example by proton NMR (Lemercinier and Jones 1996, Carbohydrate Research 296, 83-96, Jones and Lemercinier 2002, J). Pharmaceutical and Biomedical analysis 30, 1233-1247; WO 05/033 148 or WO 00/563 57). Another common method is described by Hestrin (1949) J. Biol. Chem. 180, 249-261. In some embodiments, the serotype 5 and/or 8 capsular polysaccharides of the invention are used to produce a functional antibody, such as in an animal efficacy model demonstrating antibody killing of bacteria or in a phagocytic killing assay. Bacterial measurement. Analysis of the use of monitored antibodies alone failed to demonstrate functional killing, and this analysis does not indicate the importance of Ο-acetamidine in efficacy. A capsular polysaccharide such as ash clear type 5 or 8 can be obtained directly from bacteria using a separation procedure known to those skilled in the art. See, for example, Fournier et al., (1 984) supra, Fournier et al., (1987) Households (4) "Shao (10) (10) Plant 13 8:561-567; US Patent Application Publication No. 2〇〇7/〇141〇77 And International Patent Application Publication No. W/00/56357; each of which is incorporated herein by reference in its entirety in its entirety herein in its entirety herein. In addition, it can be produced using a synthetic scheme. In addition, serotype 5 or 8 capsular polysaccharides can be produced recombinantly using genetic engineering procedures known to those of ordinary skill (see s & u et al., (1997) from 00:0/0 to 143:2395- 2405; and U.S. Patent No. 6, 〇27, 925; each of which is incorporated herein by reference in its entirety in its entirety herein. A golden yellow grape sphere that can be used to obtain a serotype 8 capsular polysaccharide. 149l59.doc -28- 201110977 _ = Staphylococcus aureus R2 pfesaq286. This strain was cultured in a modified/Frans culture medium with 10 strains of Staphylococcus aureus pfesa〇286 (American bacteria = preservation center, '· Manassas, VA; ATcc registered number (9) after hunting by flow two-cell measurement with rabbit anti- Serotype 8 polysaccharide antibodies were selected. Two populations and R2 were observed during flow cytometry. R1 and R2 were purified and recultured. R2 produced serotype 8 capsular polysaccharide. Flow cytometry analysis showed 4 @ The soil is unevenly fired. Therefore, R2 is selected to produce serotype 8 capsular polysaccharide. * It can be used to obtain isolated serotype 5 (10) and more (four) - Staphylococcus aureus strain is golden yellow grape ball (4) ESAG266. Serotype 5 capsular polysaccharides are produced during growth and yield peaks when the cells are in stationary phase. Other S. aureus Type 5 or 8 strains obtained from established preservation centers or clinical samples can be used to make the corresponding polysaccharides. Another component of the immunogenic conjugate of the present invention is a carrier molecule or protein which binds to the bacterial capsule. The term "protein carrier" and "carrier protein" means that a needle is required. Any protein molecule that binds to the antigen in the immune response of an antigen (such as a smiley membrane). The binding to the carrier enhances the immunogenicity of the antigen. The binding can be carried out according to standard procedures. The preferred protein carrier for the antigen is tetanus, diphtheria, Any mutant cross-reactive substance (CRM) of pertussis, Pseudomonas (10), Escherichia coli, Staphylococcus and Streptococcus toxins, toxoids or toxins. In one embodiment, the preferred carrier is a diphtheria toxoid CRMm derived from a strain of C. diphtheriae producing a CRM97 protein (: 7 (β197). The ATCC accession number for this strain is 149159.doc - 29-201110977 53281. A method of producing a CRMw is described in U.S. Patent No. 5,614,382, the disclosure of which is incorporated herein by reference in its entirety in its entirety in its entirety in its entirety in its entirety. Or an epitope. For example, a hapten antigen can be coupled to a bacterial toxin, a toxoid, or a T cell epitope of a CRM. See the name "Synthetic Peptides Representing a T_" as claimed on February 1, 1988.

U.S. Patent Application Serial No. 5,688, the disclosure of which is incorporated herein by reference in its entirety in its entirety in its entirety in its entirety in its entirety in its entirety in its entirety in its entirety. Other suitable carrier proteins include non-activated bacterial toxins such as cholera toxoid (e.g., as described in International Patent Application No. WO 2004/083251), Escherichia coli LT Escherichia coli ST and Pseudomonas aeruginosa aerwgkosa). Bacterial outer membrane proteins such as outer membrane complex c (OMPC), porin, transferrin binding protein, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal adhesion can also be used. Protein (psaA) or Haemophilus influenzae (10) protein D. Other proteins may also be used as carrier proteins such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tuberculin purified protein derivative (ppD). Thus, in one embodiment, the carrier protein in the immunogenic conjugate of the invention is CRMm and CRM 97 is covalently linked to the capsular polysaccharide via an amino phthalate linkage, a guanamine linkage or both. In some embodiments, the carrier protein within the immunogenic conjugate of the invention is Crm197 and CRM197 is covalently linked to the capsular polysaccharide via a thioether linkage. The carrier protein can be bound to 149159.doc • 30· 201110977 The number of lysine residues of the capsular polysaccharide can be characterized as the range of bound lysine + instance 5, in the specified immunogenic composition, CRM197 The 39 amidines may contain from 5 to 15 lysines covalently linked to the capsular polysaccharide. Another way of > number is 12% to 40°/〇(^尺)^197 is covalently attached to the capsular polysaccharide by the amine acid. For example, 'in a given immunogenic composition, CRM!9? Contains up to 22 lysines covalently linked to the capsule in 39 lysines. Another way to express this parameter is 40% to 60% CRM197 is covalently attached to the amino acid to the laughing polysaccharide. In some embodiments, CRM197 comprises 5 to 15 lysines covalently linked to cp8 in 39 amide acids. Another way to express this parameter is 12% to 4% CRMm covalently linked to the amine acid To two of the examples, CRM 丨97 contained 18 to 22 lysines covalently linked to cp5 in 39 amide acids. Another way to express this parameter is 40 /. to 60% CRM197 The amine acid is covalently attached to cp. As described above, the number of amino acid residues in the carrier protein bound to the capsular polysaccharide can be characterized as the range of bound lysine, which can be expressed as a molar ratio. In contrast, the molar ratio of bound lysine to CRM197 in the Cp8 immunogenic conjugate can range from about 18:1 to about 22:1. In one embodiment, the CP8 immunogenic conjugate is The molar ratio of the combined lysine to CRM can range from about 15:1 to about 25: 1. In one embodiment, (10) the molar ratio of bound lysine to CRMm in the immunogenic conjugate The range can be from about 14:1 to about 20:1; about 12:1 to about 18:1; about 10:1 to about 16:1; about 8,1 to about 14:1, about 6:1 to about 12 :1 ; about 4:1 to about 1 :1; about 20:1 to about 26:1 'about 22:1 to about 28:1; about 24:1 to about 30:1; about 26:1 to about 32:1; about 28:1 to about 34:1; about 30:1 to about 36:1; about 5:1 to about 149159.doc -31 - 201110977 :::1; about 5:1 to about 2〇 :1; about 10:1 to about 2:1; or about 10:1 to about 'the CP5 immunogenic conjugate, the combined lysine and 197 molar ratio may be about 3:1 to 25 In one embodiment, the molar ratio of the combined lysine to CRM, 97 in the CP5 immunogenic conjugate may range from about 5:1 to about 2: in one embodiment (2) The molar ratio of bound lysine to CRMi97 in the immunogenic conjugate may range from about "to about 20:1; from about 6:1 to about 2:1; from about 7:1 to about 2". ^; about η to about 2〇:1; about 10:1 to about 20:1; about 11:1 to about 2〇:ι; about 12:1 to about 2〇:1; about 13:1 to about 20 :1 ; 14:1 to about 2:1; about 5:ι to about 2〇·1, force 16.1 to about 20:1; about 17:1 to about 2:i; about 18:1 to about 20:1; From about 5:1 to about 18:1; about 7:1 to about 16:1; or about 91 to about 14:1. Another way of expressing the number of lytic acid residues in the carrier protein bound to the capsular polysaccharide can be in the range of binding to the lysine. For example, in a designated CP8 immunogenic conjugate, cRMi97 may comprise from 5 to 15 covalently linked to the capsular polyamine lysine in 39 lysines. Alternatively, this parameter can be expressed as a percentage. For example, the percentage of bound lysine in the designated CP8 immunogenic conjugate can range from 1% to 50%. In some embodiments, 20% to 50% of the amine acid can be covalently attached to the CP8. Alternatively, 30% to 50% CRM197 can be covalently attached to CP8 from the amine acid; 10% to 40% CRM197 lysine; 10% to 30% CRM197 lysine; 20% to 40% CRM197 lysine; 25% Up to 40% CRM197 lysine; 30% to 40. /. CRM197 lysine; 10% to 30% CRM197 lysine; 15% to 30% CRM197 lysine; 20% to 30% CRM197 lysine; 25% to 30% CRM197 amide 149159.doc -32· 201110977 Acid; 10. /. To 15°/. CRM197 is free of amine acid; or ι〇% to 12% CRM197 is covalently attached to CP8 from the amine. Further, in the designated CP5 immunogenic conjugate, CRM]97 may contain 18 to 22 lysines covalently linked to the capsule in 39 lysines. Or 'this parameter can be expressed as a percentage. For example, the percentage of bound lysine in the designated CP5 immunogenic conjugate can range from 40% to 60%. In some embodiments, 40% to 60°/. The lysine can be covalently attached to the CP5. Alternatively, 30% to 50% of CRM197 can be covalently attached to the CP5 from the amino acid; 20% to 40% of the CRMI97 is lysine; 10% to 30°/. CRM197 is from aminic acid; 50% to 70% CRM197 is from aminic acid; 35% to 65% CRM197 is from aminic acid; 30°/. Up to 60% CRM197 lysine; 25% to 55% CRMI97 from amino acid; 20% to 50. /. CRM197 lysine; 15% to 45% CRM197 lysine; 10% to 40% CRM 97 cis acid; 40% to 70% CRM197 lysine; or 45% to 75% CRM 197 covalently linked to lysine To CP5. The frequency of attachment of the capsular polysaccharide chain to the amine acid on the carrier molecule is another parameter that characterizes the combination of the capsular polysaccharide. For example, in one embodiment at least every 5 to 10 sugar repeat units of the capsular polysaccharide have at least one covalent bond between CRM!97 and the polysaccharide. In another embodiment, every 5 to 10 sugar repeating units of the capsular polysaccharide; every 2 to 7 sugar repeating units; every 3 to 8 sugar repeating units; every 4 to 9 sugar repeating units; every 6 to 11 sugar repeating units; every 7 to 12 sugar repeating units; every 8 to 13 sugar repeating units; every 9 to 14 sugar repeating units; every 10 to 15 sugar repeating units; every 2 to 6 sugar repeating units ; every 3 to 7 sugar repeating units; every 4 to 8 sugar repeating units; every 6 to 10 sugar repeating units; each to 1] L sugar repeating units; every 8 to 12 sugar repeating units; every 9 Up to 13 sugar repeat units; each (7) to the bucket 149159.doc -33- 201110977 sugar repeat unit; every 10 to 20 sugar repeat units; or every 5 to 1 sugar repeat unit exists at least one between CRM, Covalent bond between 97 and polysaccharide. In another embodiment, each of the capsular polysaccharides is 2, 3, 4, 5, 6, 7, 8, 9, 1 〇, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 The sugar repeating unit has at least one bond between the CRMm and the capsular polysaccharide. One embodiment of the present invention provides an immunogenic composition comprising any immunogenic conjugate comprising a Staphylococcus aureus 5 or 8 capsular polysaccharide in combination with the above carrier protein. The term "immunogenic composition" relates to any pharmaceutical composition containing an antigen, such as a microorganism or a component thereof, which composition can be used to elicit an immune response in an individual. The immunogenic compositions of the invention can be used to protect or treat humans susceptible to S. aureus (administering an immunogenic composition by means of a systemic 'epidermal or mucosal route), or can be used to generate passives that can be used to confer another body An immunological multi-plant or monoclonal antibody preparation. Such administration may include injection via the intramuscular, intraperitoneal, intradermal or subcutaneous route; or administration to the oral/digestive, respiratory or genitourinary tract via the mucosa. In one embodiment, intranasal administration is used to treat or prevent the nasopharyngeal band of S. aureus' to reduce infection at its initial stage. The immunogenic composition can also be used to produce a functional antibody, as measured in an animal potency model or via a conditioned phagocytic killing assay, based on killing the bacteria. The optimal amount of the components of a particular immunogenic composition can be determined by standard studies' including the appropriate immune response of the observed individual. After the initial vaccination, the individual can receive one or several adequate intervals of booster immunization. The immunogenic compositions of the invention may also include one or more of the following antigens: 149159.doc • 34- 201110977

ClfA, ClfB, SdrC, SdrD, SdrE MntC/SitC/Saliva-binding protein, IsdB, IsdA, Opp3a, DltA, HtsA, LtaS, SdrH, SrtA, SpA, SBI, a-hemolysin (hla), β-hemolysin, Fibronectin binding protein A (fnbA), coagulase, map, Panton-Valentine leukocyte (pvl), gamma toxin (hlg), ica, immunodominant ABC transporter, RAP, autolysin, laminin Receptor, isaA/pisA, IsaB/pisB, SPOIIIE, SsaA, EbpS, SasF, SasH, EFB (FIB), FnbB,

Npase, EBP, osteophyte-binding protein n, aureolysin precursor (AUR)/Seppl, Cna, TSST-1, mecA, dPNAG, GehD, EbhA, EbhB, SSP-1, SSP-2 HBP, vitronectin binding protein, HarA, enterotoxin A, enterotoxin sputum, enterotoxin Cl, and novel autolysin. In one embodiment, the immunogenic composition of the present invention further comprises an adjuvant, a buffer, a cryoprotectant, a salt, a second-order cation, a non-ionic clearing agent, a radical oxidation inhibitor, a diluent or a carrier. At least one of them. In one embodiment, the adjuvant in the immunogenic composition of the invention is an aluminum based adjuvant. In one embodiment, the adjuvant is an aluminum-based adjuvant selected from the group consisting of aluminum lanthanum aluminum phosphate and aluminum hydroxide. In one embodiment the adjuvant is aluminum phosphate. Can enhance the immune response when administered together

149159.doc Adjuvant is a substance when used with an immunogen or antigen. Many cytokines or lymphocytes 1 -35- 201110977 18 (and their mutant forms); interferon-α, β and γ; granule macrophage community stimulating factor (GM-CSF) (see, for example, U.S. Patent No. 5,078,996 and ATCC accession number 39900); macrophage community stimulating factor (M-CSF); granule globule community stimulating factor (G-CSF); and tumor necrosis factor alpha and beta. Other adjuvants that can be used with the immunogenic compositions described herein include chemokines including, without limitation, MCP-1, ΜΙΡ-1α, MIP-Ιβ, and RANTES; adhesion molecules, such as selectins, for example L-selectin, Ρ-selectin and Ε-selectin; mucin-like molecules such as CD34, GlyCAM-1 and MadCAM-1; integrin family members such as LFA-1, VLA-1, Mac-Ι and pi 50.95; immunoglobulin superfamily members such as PECAM, ICAM (eg ICAM-1, ICAM-2 and ICAM-3), CD2 and LFA-3; costimulatory molecules such as B7-1, B7-2, CD40 and CD40L Growth factors, including vascular growth factor, nerve growth factor, fibroblast growth factor, epidermal growth factor, PDGF, BL-1, and vascular endothelial growth factor; receptor molecules, including Fas, TNF receptor, Fit, Apo-1 , p55, WSL-1, DR3, TRAMP, Apo-3 'AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2 and DR6; and Caspase, including ICE. Adjuvants suitable for enhancing the immune response may further include, without limitation, MPLTM (3-0-deacetylated monophosphonyl lipid A, Corixa; Hamilton, MT), which is described in U.S. Patent No. 4,912,094 . Also suitable as adjuvants are synthetic lipid A analogs or aminoalkyl glucosamine phosphate compounds (AGP) or derivatives or analogs thereof available from Corixa, and those described in U.S. Patent No. 6,113,918. Such AGP is 2- 149159.doc -36- 201110977 [(R)-3 -tetradecyloxytetrakisylamino]ethyl 2_deoxy_4_〇_phosphinyl-3-0 -[(R)-3-tetradecyloxytetradecyl]_2_[(R)_3tetradecyloxytetradecyl-amino]-bD-grape sigma sucrose, Also known as 529 (formerly known as RC529). This 529 adjuvant is formulated into an aqueous form (AF) or a stable emulsion (SE). Other adjuvants include cell wall-derived peptides such as N-ethyl thiol-cell wall total _l-threonyl-D-iso branide (thr-MDP), N-ethyl thiol-deciding wall Indenyl _ L-alanine-2-(Γ-2'-bis(hexadecylglyceryl_3_hydroxyphosphonyloxy)-ethylamine (ΜΤΡ-ΡΕ); oil-in-water emulsion 'such as MF59 (U.S. Patent No. 6,299,884) (containing 5 ° / squalene, 〇 5 ° / 〇 polysorbate 8 〇 and ο %% Span 85 (depending on the amount of MTP-PE), using microfluidics Chemical instrument such as 110Y microfluidizer (Microfluidics, Newton, MA) formulated into submicron particles) and SAF (containing 10% squalene, 0.4% polysorbate 80, 5 ° / oxidized isopropylene block Copolymer l 121 and thr-MDP, microfluidized into submicron emulsion or vortexed to produce emulsion of larger particle size); incomplete Freund's adjuvant (IFA); aluminum salt (矾)' Aluminum hydroxide, aluminum phosphate, aluminum sulfate; Amphigen; Avridine; L121/squalene; D-propionate-polylactide/glycoside; oxidized isopropenol polyol; Killed Bode Saccharomyces (5orc?eie//a); saponins, such as those described in US Patent No. 5, 〇57, 540, StimulonTM QS-21 (Antigenics, Framingham, MA), US Patent No. 5,254,339 (isconlatrix®) CSL Limited, Parkville, Australia) and immunostimulating complex (ISC〇MS); Mycobacterium tuberculosis; bacterial lipopolysaccharide; synthesis 149159.doc -37- 201110977 polynucleotides such as oligonucleotides containing CpG motifs (e.g., U.S. Patent No. 6,207,646), IC-31 (Intercell AG, Vienna, Austria) 'Full in European Patent Nos. 1,296,713 and 1, 3 26, 63 4; pertussis toxin (PT) or Mutant, cholera toxin or a mutant thereof (e.g., U.S. Patent Nos. 7,285,281, 7,332,174, 7,361,355 and 7,3,84,640), or Escherichia coli, not to a thermotoxin (lt) or a mutant thereof, 1/1'-1^63, 1^1-1172 (for example, U.S. Patent No. 6,149,919, '7'115, 73 0 and 7,291,588). The immunogenic composition may include medicine as appropriate. An acceptable carrier. The term "pharmaceutically acceptable carrier" means approved for use by animals, including humans and non-human mammals, by a federal regulatory agency, state government, or other regulatory agency or listed in the US Pharmacopoeia or other generally recognized pharmacopoeia. Carrier. The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which the pharmaceutical composition is administered. Water, physiological saline solution and dextrose aqueous solution and glycerol solution can be used as a liquid carrier, especially for injectable solutions. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. w Martin. The formulation should be suitable for the mode of administration. The immunogenic composition of the present invention may further comprise one or more other "immunomodulators" which are agents which interfere with or alter the immune system' thereby observing humoral and/or cell-mediated immune upregulation or Down. In one embodiment, a bodily fluid and/or cell mediated immune system branch upregulation is provided. Examples of certain immunomodulatory agents include, for example, the adjuvants or the menopause hormones or Isc〇matrix® (CSL Limited; Parkville, Australia) </ RTI> 49159. doc • 38-201110977, especially in U.S. Patent No. 5,254,339. Non-limiting examples of adjuvants that can be used in the immunogenic compositions of the invention include the Magic 61 adjuvant system (Ribi lnc.; Hamilton, MT), hydrazine, mineral gels (such as aluminum hydroxide gel), water Oil-in-water emulsion, water-in-oil emulsion (such as complete Freund's adjuvant and incomplete Freund's adjuvant), block copolymer ((^1^;八^,〇八), (^-21 (Cambridge Biotech Inc) .; Cambridge, ΜΑ) 'SAF-M (Chiron; Emeryville, CA), Amphigen® adjuvant, saponin, Quii A or other saponin fraction, monosulfonyl lipid a and aflide lipid amine adjuvant. Non-limiting examples of oil-in-water emulsions in the immunogenic compositions of the invention include modified SEAM 62 and SEAM 1/2 formulations. Modified SEAM 62 is 50/〇 (v/v) squalene (Sigma), I〇/〇(v/v)Span® 85 Cleaner (ICI Surfactants), 7%.7% (v/v) Polysorbate 80 Cleaner (ICI Surfactants), 2.5% (v/v) Ethanol, 200 Oil-in-water emulsion of mcg/ml Quil A, 100 mcg/ml cholesterol and 〇·5% (ν/ν) lecithin. Modified SEAM 1/2 is 5% (ν/ν) squalene, i% (v/v)span® 85 cleaner, 0·7 /〇(ν/ν) polysorbate 8〇 detergent, 2.5% (v/v) ethanol, 100 mcg/ml Quil A and 50 mcg/ml cholesterol oil-in-water emulsion. Can be included in immunogenic combinations Other "immunomodulators" in the context include, for example, one or more interleukins, interferons or other known cytokines or chemokines. In one embodiment, the adjuvant may be a cyclodextrin derivative or a polyanionic Polymers, such as those described in U.S. Patent Nos. 6,165,995 and 6,610,310, respectively, it being understood that the immunomodulatory agents and/or adjuvants used will be administered to the individual to which the immunogenic composition is administered, the route of administration. And the number of injections. In addition to a plurality of staphylococcal capsular polysaccharide-protein conjugates, the immunogenic composition of the present invention 149159.doc-39·201110977 may further comprise one or more preservatives. The biological products in the dose bottles contain preservatives with a few exceptions. Vaccine preparations containing preservatives include benzethonium chloride (anthrax), 2_phenoxyethanol (DTaP, HepA, Lyme, P〇li〇 (non- Intestine)), phenol (pneumonia, typhoid fever (non- Intestine), Vaccinia) and thimerosal (DTap, DT, Tcj,

Vaccines of HePB, Hib, influenza, JE, meningitis, pneumonia, rabies. Preservatives approved for injectable pharmaceuticals include, for example, chlorobutanol, m-nonylphenol, methylparaben, propylparaben, 2-phenoxyethanol, sulphonium, chlorine Ammonium benzoate, benzoic acid, benzyl alcohol, phenol, thimerosal, and stearic mercury. The formulations of the present invention may further comprise one or more of the following: buffers, salts, second-order cations, nonionic detergents, cryoprotectants such as sugars, and antioxidants such as free radical scavengers or integrators, or Any multiple combination thereof. The choice of any component, such as a chelating agent, may determine if another component (e.g., a scavenger) is required. The final composition to be administered for administration should be sterile and/or pyrogen free. Those skilled in the art will be able to determine, empirically, which combination of these and other components are preferred in the immunogenic compositions of the present invention containing the preservative, depending on various factors, such as the particular storage and administration conditions necessary. In certain embodiments, formulations of the invention that are compatible with parenteral administration comprise - or a plurality of physiologically acceptable buffers selected from, but not limited to, (trimethylamine), disc salts , acetate, acid salt, citrate, glycine, histidine and succinate. In certain embodiments, the formulation is buffered to a positive range of from about 6.0 to about 9.0, preferably from about 6.4 to about 74. 149159.doc 201110977 In certain embodiments, the devaluation of the Hutang I$α &amp; or the formulation is too: The immunogenic combination of the invention Intraoperative...! The pH of the formulation can be adjusted using this technique. The pH of the formulation can be adjusted to 3_〇 to 8.0. In some embodiments, the positive value of the formulation can be or can be adjusted to 3 至 to 6·〇, 4.0 to 6.0, or 5 to 8 〇. In other embodiments, the positive value of the formulation can be or can be adjusted to about 3 〇, about 3.5, about 4. 〇, about 4 5, about 5.00, about 5.5, about 5.8, about 6 〇, about 65, about 7 〇, about η or about 8 〇. In some embodiments, the value of ρΗ can be or can be adjusted to 4 5 to Μ, or 4, 5 to 6, 5, 5·〇 to 5, 4, 5 4 to 5 5, 5 ... 6, 5 to 5 77 8 5.8 to 5_9, 5_9 to 6.0, 6.0 to 6.1, 6.1 to 6.2, 6.2 to 6'3 to 6-5, 6'5 to 7.0, 7.0 to 7.5 or 7.5 to 8.0. In a particular embodiment, the formulation has a pH of about 5 8 ^. In certain embodiments, the formulation of the invention that is compatible with parenteral administration comprises one or more divalent cations including, but not limited to, MgCh, CaCh and

MnCl2 has a concentration ranging from about 〇 j mM to about 1 mM, preferably up to about 5 rnM. In certain embodiments, formulations of the invention that are compatible with parenteral administration comprise one or more salts including, but not limited to, sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate, the salts being non- Physiologically acceptable ionic concentrations are present when administered intra-intestinal to an individual and are included in the final formulation at a final concentration that produces a selected ionic strength or volume osmotic concentration. The final ionic strength or osmotic pressure of the formulation will be determined by multiple components, such as ions from buffer compounds and other non-buffering salts. Preferably, the salt NaCH is present in the range of up to about 250 mM, and the salt concentration is selected to be complementary to other components (e.g., sugar) such that the final total volume osmotic concentration of the 149159.doc 201110977 is formulated with parenteral administration (e.g., intramuscular). Or subcutaneous injection) is compatible and will promote long-term stability of the immunogenic component of the immunogenic composition formulation over a wide range of temperatures. The salt-free formulation will allow for an increase in the range of one or more selected cryoprotectants to maintain the desired final volumetric osmotic concentration level. In certain embodiments, a formulation of the invention that is compatible with parenteral administration comprises one or more cryoprotectants, which are selected from, but are not limited to, disaccharides (eg, lactose, maltose, sucrose, or trehalose) and Poly-based hydrocarbons (eg, galactitol, glycerol, mannitol, and sorbitol).

In certain embodiments, the volumetric osmolality of the formulation is in the range of from about 2 〇〇 m〇s/L to about 800 m〇s/L, preferably from about 25 〇m〇s/L to about 500. mOs/L, or about 300 mOs/L to about 400 mOs/L·. The salt-free formulation may contain, for example, from about 5% to about 25% sucrose, and preferably from about 7% to about 15% or from about 1% to about 12% sucrose. Alternatively, the salt-free formulation may contain, for example, from about 3% to about 12% sorbitol, and preferably from about 4% to about 7 ❶/❶ or from about 5% to about 6% sorbitol. If a salt such as sodium chloride is added, the effective range of sucrose or sorbitol is relatively lowered. These and other osmotic pressure and volumetric osmotic concentration considerations are well within the skill of the art. In certain embodiments, the formulations of the invention that are compatible with parenteral administration comprise one or more free radical oxidation inhibitors and/or chelating agents. Various free radical scavengers and chelating agents are known in the art and are useful in the formulations and methods of use described herein. Examples include, but are not limited to, ethanol, EDTA, EDTA/ethanol combination, triethanolamine, mannitol, histidine, glycerol, sodium citrate, phytate, tripolyphosphate, ascorbic acid/ascorbic acid 149159 .doc -42· 201110977 acid salt, succinic acid / succinate, malic acid / cis-succinate, desferal, EDDHA and DTPA ' and different combinations of the two or more . In certain embodiments, at least one non-reducing free radical scavenger can be added at a concentration effective to enhance the long-term stability of the formulation. - or a plurality of free radical oxidation inhibitors/chelators may also be added in various combinations, such as scavengers and second order cations. The choice of chelating agent will determine if a scavenger is required. In certain embodiments, formulations of the invention that are compatible with parenteral administration comprise one or more nonionic surfactants including, but not limited to, polyoxyethylene sorbitan fatty acid esters, polysorbates _8〇 (Tween 8〇), Polysorbate-60 (Tween 60), Polysorbate-40 (Tween 40) and Polysorbate-20 (Tween 20); Polyoxyethylene alkyl ethers, including (but not limited to) Bnj 58, Brij 35, and other surfactants, such as Trh〇n χ _ 100, Tnton Χ-114, ΝΡ40, Span 85, and oxidized isopropylene series nonionic surfactants (eg, oxidized isopropene 121) Preferably, the concentration of the preferred component polysorbate _80 is from about 0.001% to about 2. /. The concentration of (up to about 0.25 Å/min.) or polysorbate _4 为 is from about 1% to about 1% (up to about 5% is preferred). In certain embodiments, the formulations of the present invention comprise one or more other stabilizers suitable for non-aged techniques, such as a reducing agent comprising at least one thiol (-SH) group (eg, cysteine, N-B Mercaptocysteine, reduced glutathione, sodium thioglycolate, thiosulfate, monothioglycerol or mixtures thereof). Alternatively, or as the case may be, the preservative-containing immunogenic composition formulation of the present invention may be further stabilized by the removal of oxygen from a storage container that protects the formulation from light (e.g., by using a brown glass container). 149159.doc • 43· 201110977 The preservative-containing immunogenic composition formulation of the present invention may comprise one or more pharmaceutically acceptable carriers or excipients, including any excipients that do not induce an immune response by themselves. . Suitable excipients include, but are not limited to, macromolecules such as proteins, sugars, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose (Pa〇letti et al., 2, 1, 19) : 2 11 8), trehalose, lactose and lipid aggregates (such as oil droplets or liposomes). 3 Xuan et al. are well known to those skilled in the art. Pharmaceutically acceptable excipients are discussed, for example, in Gennaro, 2, Remingt〇n: The composition of the invention; or as a solution or suspension of water (iv). The liquid formulation is preferably administered directly from its packaged form and, as such, is preferred for injections that do not require reconstitution in an aqueous medium as required by the lyophilized compositions of the present invention. Direct delivery of an immunogenic composition of the invention to an individual can be administered parenterally (intramuscularly, intraperitoneally, intradermally, intradermally, intravenously, or in the interstitial space of a tissue or transrectally or orally). , local, transdermal, intranasal, ocular, aural, pulmonary or other mucosal administration. In a preferred embodiment, parenteral administration is by intramuscular injection, for example, administration of an individual's upper arm. It may be via a needle (for example a hypodermic needle), but alternatively = needle-free injection. A typical intramuscular dose is 〇5 mL. The composition of the invention is in various forms, for example a liquid solution or suspension for injection. The composition may be prepared as a powder or spray for administration via the lung, for example, in the presence of 55 tb + *4·, in other embodiments, the composition may be prepared as a suppository or pessary or By nasal, aural or ocular administration, for example, a spray, 149159.doc • 44 - 201110977 drops, gel or powder. The amount of conjugate in each immunogenic composition dose is selected to induce an immunoprotective response. No significant amount of adverse reactions. This amount can be seen in Portugal It is determined by the serotype of the bacterium. In general, each dose will comprise 〇1 to 1 〇〇 polysaccharide, especially 0.1 to 10 μ § polysaccharide, and more particularly! to 5 盹 polysaccharide. Components of specific immunogenic compositions The optimal amount can be read by standard studies to include the appropriate immune response of the observed individual. After the initial vaccination, the individual can receive one or more sufficiently spaced booster immunizations. Packaging and Dosage Forms The immunogenic compositions of the invention can be packaged. In unit or multi-dose form (eg 2, 4 or more). For multi-dose forms, vials are usually, but not necessarily, better than pre-filled syringes. Suitable multi-dose forms include (do not: to 1 dose) / Container '.. (1) cc / iv in some embodiments 'agents 〇 · 5 mL dose. See, for example, International Patent Application No. 2/7/127, 668, the entire disclosure of which is incorporated herein by reference. The composition may be present in a small or other suitable storage container or may be present in a pre-filled delivery device, such as a one-component or vacant syringe that may be provided with or without a needle. The syringe typically (but not necessarily) contains Single dose The invention may also envisage multi-dose pre-filling multiple doses. The same m' vial may include a single dose, but may alternatively be included, but in a typical injection of the composition, the dose Formulated for administration and administration to an adult, cyan effective dosage volume can be determined in a conventional manner with a dose of 0.5 mL volume. In certain embodiments human subjects. In certain embodiments, the dose I49l59.doc •45· 201110977 A juvenile, youth, infant or infant (i.e., no more than one year old) human individual, and in preferred embodiments, can be administered by injection. The liquid immunogenic composition of the present invention is also suitable for use in a reconstituted lyophilized form. Other immunogenic compositions. Where the immunogenic composition is used in such temporary reconstitution, the invention provides for having two or more vials, two or more pre-filled syringes or each one Or a plurality of sets wherein the syringe contents are used to restore the contents of the vial prior to injection, or vice versa. Alternatively, 'the immunogenic composition of the invention can be lyophilized and reconstituted, for example, using one of many freeze-drying methods well known in the art to form dry, regular (eg, spherical) particles, such as pellets or microspheres, The particle characteristics (such as the average diameter size) can be controlled by controlling the specific preparation method to control the immunogenic composition, and can further comprise particles which are optionally prepared or contained in separate dry, regular (eg, spherical) shapes (such as micro An adjuvant in a pellet or microsphere). In these embodiments, the invention further provides an immunogenic composition kit comprising: a first group comprising a stabilized, dried immunogenic composition and, optionally, one or more preservatives of the invention And a second component comprising a sterile aqueous solution for reconstituting the first component. In certain embodiments, the aqueous solution comprises one or more preservatives and optionally at least one adjuvant (see, e.g., w〇 2〇〇9/109550', which is incorporated herein by reference). In still another embodiment, the multi-dose form of the container is selected from, but not limited to, a group consisting of: - a laboratory glass orphan, a flask, a beaker, an S: tube, a vinegar , bioreactors, conduits, tubing, 149159.doc -46· 201110977 garments, jars, vials, vials (eg (eg rubber stoppers, screw caps), ampoules, main emitters, double chambers or Multi-chamber injection of pregnant gold ΐΛ 益 益 主 main plug, syringe plunger, rubber cover, plastic cover, glass u 把 塥盍 塥盍 乐, music tube and disposable pen and the like. The container of the present invention is not subject to The material is limited and includes materials such as glass, gold paste such as steel, residual steel, (iv), and polymers (e.g., thermoplastic polymers, elastomers, thermoplastic elastomers). In a particular embodiment, the container of this type is a 5-machine 8-phase summer glass bottle with a butyl rubber stopper. Those skilled in the art will appreciate that the above-described forms are not exhaustive, but merely provide those skilled in the art with a variety of materials that can be utilized by the present invention. Other forms covered by the present invention can be found in the public catalog of laboratory equipment suppliers and manufacturers, such as υη_

States Plastic Corp. (Lima, OH), VWR. Methods of Making Immunogenic Conjugates The present invention also encompasses methods of making the immunogenic conjugates described herein. A method for making an immunogenic conjugate of the invention comprises covalently binding a capsular polysaccharide to a carrier protein using a binding chemistry, the binding chemistry comprising CDI (1,1-monodiimidazole), CDT (1,1) - carbonyl-dioxa-4-triazole) or PDPH (3 - (2_ ° ratio. thiodithio)). Thus, one embodiment of the present invention provides a method for producing an immunogenic conjugate based on Cdt, which comprises a combined S. aureus serotype 5 or 8 capsular polysaccharide and a carrier protein, the method comprising the following steps : a) combining S. aureus serotype 5 or 8 capsular polysaccharide with imidazole or triazole to produce a compounded polysaccharide; b) bringing the compounded polysaccharide and CDT in an organic solvent and from about 0.1% to about 0.3% w/v Reacts in water to produce activated serotypes 5 149159.doc 47· 201110977 or 8 inch membranes to purify ") purified activated serotypes 5 or 8 capsules to produce purified activated serotype 5 or 8 capsules; The purified activated serotype 5 or 8 masking membrane multi-enzyme is reacted with the carrier protein in an organic solvent to produce a serotype 5 or 8-inch membrane: a carrier protein conjugate; and e) hydrolyzed gold-clearing type 5 or 8 inches Membrane _ : carrier protein conjugate, removing unreacted reactive groups; thereby producing an immunogenic conjugate comprising S. aureus serotype ... capsular scorpion and carrier protein. In an embodiment In, before step (4) Purification of the activated serotype 5 or 8 membranous cholestases into a carrier protein. In one embodiment of the invention, another CDT-based method of making an immunogenic conjugate is provided, the conjugate comprising a combined gold Staphylococcus aureus serotype 5 or 8 membranes to listen to the carrier protein, the method comprises, as in step I. a) S. aureus serotype 5 or 8 capsular polysaccharide and imidazole or three. Sit combination to produce a compound polysaccharide; b) reacting the combined vinegar with CDT in an organic solvent (M. / to about 3% 3% w/v water to produce an activated serotype or 8 capsular polysaccharide; c) to activate the serotype 5 or 8 capsular polysaccharide and carrier egg substance are reacted in an organic agent to produce serotype 5 or 8 capsular polysaccharide: carrier protein conjugate; and d) hydrolyzed serotype 5 or 8 capsular polysaccharide: carrier protein conjugate, removed An unreacted reactive group; thereby producing an immunogenic conjugate comprising a S. aureus serotype 5 or 8 serotonin polysaccharide in combination with a carrier protein. In one embodiment, a CDT-based immunogenic binding is produced. The organic solvent in the method of the object is a polar aprotic solvent. In one embodiment, the organic solvent is selected from the group consisting of disulfoxide (DMSO), dimethylformamide 149l59.doc -48- 201110977 (DMF), dimethylacetamide, N a linear aprotic solvent consisting of a group consisting of pyrrolidone and hexamethylphosphonium (HMPA). In one embodiment, the organic solvent is DMSO. In one embodiment, In the method of making an immunogenic conjugate based on CDT, the step of reacting the conjugated polysaccharide with CDT comprises providing a cdt that is about 20 times molar excess compared to how cool. In one embodiment, the immunogen is produced on a CDT-based basis. In the method of sex conjugate, the step of purifying the activated serotype 5 or 8 capsular polysaccharide comprises a dialysis transition. In one embodiment, the carrier protein within the method of making an immunogenic conjugate based on CDT is CRMm. In one embodiment, the activated serotype 5 or 8 capsular polysaccharide in the method of making the immunogenic I1 conjugate is reacted in a weight ratio of about 1:1. In one embodiment, in the method of making an immunogenic conjugate based on CDT, the step of hydrolyzing the serotype 5 or 8 polysaccharide: carrier protein conjugate to remove unreacted reactive groups comprises diluting in a buffer And at about 2 〇 it to about 26. Maintain a positive value of about 8, 8 to about 92 for at least 4 hours. In one embodiment, the step of hydrolyzing the serotype 5 or 8 capsular polysaccharide:carrier protein conjugate comprises diluting in a buffer and maintaining a positive value of about &quot;about&quot; for about 4 hours. In one embodiment, the serotype 5 or 8 serotonin: carrier protein complex produced according to the CDT-based method for producing immunogenic compounds is purified. In one embodiment, the purified serotype 5 or 8 inch membrane polysaccharide: carrier protein conjugate comprises diafiltration. 149159.doc -49· 201110977 In one embodiment, in a method of making an immunogenic conjugate based on CDT, the serotype 5 or 8 polysaccharide is lyophilized and resuspended prior to reacting the conjugated polysaccharide with CDT. In one embodiment, the conjugated polysaccharide and the carrier protein are separately lyophilized and resuspended prior to reacting the conjugated polysaccharide with CDT. In one embodiment, the lyophilized compounded polysaccharide and/or lyophilized carrier protein is resuspended in an organic solvent. In one embodiment, the organic solvent is DMSO. In one embodiment, in a method for producing an immunogenic conjugate based on Cdt, the activated serotype 5 or 8 capsular polysaccharide is combined with a carrier protein, and the purified activated serotype 5 or 8 capsular polysaccharide is The carrier proteins were lyophilized and resuspended in the ocean. In one embodiment, the carrier protein is CRMm and the CRMw is diafiltered against NaCI prior to lyophilization. In one embodiment, the weight ratio of NaCi/cRM is adjusted to about 〇·5 to about 丨5 relative to NaC1 prior to lyophilization and the weight ratio of NaCi/cRM is adjusted. One embodiment of the present invention provides a basis for A method of making an immunogenic conjugate of PDPH comprising a combined S. aureus serotype 5 or 8 capsular polysaccharide and a carrier protein, the method comprising the steps of: a) causing S. aureus serotype 5 Or 8 capsular polysaccharides are reacted with stone and imine in an organic solvent to produce a pDpH-linked polysaccharide; b) reacting the PDPH-linked polysaccharide with a reducing agent to produce an activated polysaccharide; 8 capsular polysaccharide, producing purified activated serotype 5 or 8 inch membrane brewing; d) reacting carrier protein f with acetic acid in organic solvent to produce activated carrier protein; e) purifying activated carrier protein, producing purification Activating the carrier protein; f) reacting the purified activated serotype 5 or 8 I49159.doc -50- 201110977 capsular polysaccharide with the purified activated carrier protein to produce a serotype 5 or 8 capsular polysaccharide: carrier protein conjugate; g) Hydrolyzing serotype 5 or 8 capsular polysaccharide: carrier protein conjugate, removing unreacted reactive groups; thereby producing immunogenic binding comprising S. aureus serotype 5 or 8 capsular polysaccharide in combination with carrier protein Things. In one embodiment, the bromoacetic acid used in the method of making an immunogenic conjugate based on PDPH is 7V-hydroxybutylimine bromoacetate (BAANS). In one embodiment, the carrier protein used in the PDPH-based method of the invention is CRM197 and the BAANS is added at a CRM197:BAANS weight ratio of from about 1:0.1 to about 1:0.5. In one embodiment, the organic solvent within the method of making an immunogenic conjugate based on PDPH is a polar aprotic solvent. In one embodiment, the organic solvent is a polar aprotic solvent selected from the group consisting of DMSO, DMF, dimethylacetamide, JV-mercapto-2-pyrrolidone, and HMPA. In one embodiment, the organic solvent is DMSO. In one embodiment, the carbodiimide used in the method of making an immunogenic conjugate based on PDPH is 1-ethyl-3-(3-didecylaminopropyl)-carbodiimide ( EDAC). In one embodiment, the step of reacting the serotype 5 or 8 capsular polysaccharide with PDPH and EDAC in an organic solvent comprises maintaining the polysaccharide: PDPH: EDAC in a weight ratio of about 1:5:3. In one embodiment, the reducing agent used in the method of making an immunogenic conjugate based on PDPH is dithiothreitol (DTT). In one embodiment, in the method of making an immunogenic conjugate based on PDPH, the steps of purifying the activated serotype 5 or 8 capsular polysaccharide and purifying the carrier egg 149159.doc • 51 - 201110977 white matter each comprise diafiltration. In one embodiment, the carrier protein within the method of making an immunogenic conjugate based on PDPH is CRMm. In one embodiment, in the method of making an immunogenic conjugate, the activated serotype 5 or 8 capsular polysaccharide is reacted with CRMm in a weight ratio of about 1:1. In one embodiment, in the method of producing an immunogenic conjugate based on PDPH, the step of hydrolyzing a serotype 5 or 8 polysaccharide: carrier protein conjugate to remove unreacted reactive groups comprises adding a cysteamine salt Acid salt. In one embodiment, the serotype 5 or 8 smiley polysaccharide: carrier protein conjugate produced according to the PDPH-based method of making an immunogenic conjugate is purified. In one embodiment, the purified serotype 5 or 8 capsular polysaccharide:carrier protein conjugate comprises diafiltration. In one embodiment, in the method of making an immunogenic conjugate based on PDPH, the purified activated polysaccharide and purified are purified prior to reacting the purified activated serotype 5 or 8 capsular polysaccharide with the purified activated carrier protein. The activated carrier protein was lyophilized and resuspended. In one embodiment, the lyophilized activated polysaccharide and/or lyophilized activated carrier protein is resuspended in an organic solvent. In one embodiment, the organic solvent is Dms. As used herein, "lyophilized" means a dehydration process in which the peripheral pressure is reduced while freezing the bacterial capsular polysaccharide to allow the frozen water to sublimate from the solid phase to the gas phase. Any method of cold-drying sugar known in the art can be used. See, for example, Harris and Angal (1989) "Protein Purification Methods", Kennedy and Cabral, ed. "Recovery Processes for Biological Materials" (John I49159.doc -52- 201110977

Wiley &amp;Sons;1993); U.S. Patent No. 4,134,2, 14, and International Patent Application Publication No. WO 2003/086471; each of which is incorporated herein by reference in its entirety herein in its entirety. A cryoprotectant such as sucrose, glucose, lactose, trehalose, arabinose, xylose, galactose, sorbitol or mannitol may optionally be included during lyophilization. As used herein, &quot;activated&quot; means that the bacterial membrane is multi-riding or carrier protein in such a way as to facilitate its incorporation (i.e., at least a portion must be capable of binding the mussel to the carrier molecule) for repair. For example, for the CDT-based binding method of the present invention, the polysaccharide is activated in a low moisture environment (for example, in DMSO) to form a triazolyl phthalate moiety having a hydroxyl group and a sulfhydryl group having a carboxylic acid. Azole moiety. The activated polysaccharide can then be reacted with the CRM丨μ protein to subject the amino acid residue in CRM97 to nucleophilic displacement of the triterpenoid and form a carbaryl linkage (for the active thiol) and a stilbene linkage (for Active acid retardant). In contrast, for the PDPh-based binding method of the present invention, both the carrier protein and the polysaccharide are activated prior to binding: activation of Crm197 includes reaction of an amine group with N-hydroxybutylimine of bromoacetate. The bromoethenyl group is introduced into the CRMm protein; and 2) the activation of the polysaccharide comprises the ruthenium ruthenium group and the sulfhydryl group which are activated by the carbodiimide of the N-ethyl decylamino mannitol in the polysaccharide. The doped difunctional linker pDpHi thiol couple was then reduced with DTT. The activated polysaccharide can then be reacted with the activated carrier protein to react the thiol of the polysaccharide thiolated with PDPH with the ethidium bromide group of the activated carrier protein to form a covalent thioether bond by bromine displacement. According to the method of the present invention, capsular polysaccharide, carrier protein and/or poly--protein conjugate can be purified. Any method known in the art for purifying I49159.doc •53-201110977 polysaccharides or proteins, such as concentration/diafiltration, precipitation/dissolution, column chromatography and depth filtration, can be used. See, for example, FarrSs et al. (1996) B/oiec/mo/· 10:375-380; Gongalves et al., Communicating

Current Research and Educational Topics and Trends in Applied Microbiology (Antonio Mendez Vilas, ed. 0, 1st edition. Badajoz, Espanha: Formatex; 2007. pp. 450-457); Tanizaki et al., (1996) */. Mz'eroMo/, 27:19-23; and U.S. Patent No. 6,146,9, 2; and U.S. Patent Application Publication No. 2008/0286838; each of which is incorporated herein by reference in its entirety herein in its entirety. As used herein, the term "isolated" or "purified" means that a substance is removed from its original environment (eg, its natural environment in its natural presence, or removed from its host organism when it is a recombinant entity, or Moved from one environment to a different environment). For example, an isolated polysaccharide, peptide or protein is substantially free of cellular material or other contaminating protein of the cell or tissue source from which the protein is derived' or when chemically synthesized or present in a mixture as part of a chemical reaction, It does not contain chemical precursors or other chemicals. In the present invention, the protein or polysaccharide may be detached from bacterial cells or cell debris, and thus it is provided in a form suitable for the manufacture of an immunogenic composition. The term "isolated" may include purification, including, for example, a method of purifying a capsular polysaccharide as described herein. The phrase "substantially free of cellular material" includes the preparation of polysaccharides/polypeptides/proteins in which the polysaccharide/polypeptide/protein is isolated from the cellular components of cells which are separable or recombinantly produced. Thus, polypeptides/proteins f, polysaccharides or conjugates substantially free of cellular material or other compounds include preparations having less than about 30°/. 20%, 10%, 5%, 2.5% or 1% (by dry weight) of peptide/protein, multi-St or conjugate of contaminating protein, polysaccharide or other compound. When the polypeptide/protein is produced recombinantly, it preferably also contains substantially no culture medium, i.e., the medium represents less than about 20%, 10%, 5%, 4%, 3%, 2% or 1% of the protein preparation volume. . When a polypeptide/protein or polysaccharide is produced by chemical synthesis, it preferably does not substantially contain a chemical precursor or other chemical, i.e., it is separated from a chemical precursor or other chemical involved in the synthesis of the protein or polysaccharide. Thus, in addition to the relevant polypeptide/protein or polysaccharide fragment, the polypeptide/protein or polysaccharide preparations also have less than about 30%, 20%, i〇〇/Q, 5%, 4%, 3%, 2% or 1%. A chemical precursor or compound (on a dry weight basis). The immunogenic conjugates produced by any of the methods described herein can be stored in water or low ionic strength neutral pH buffer or lyophilized to a dry powder. Methods of Inducing an Immune Response and Preventing S. aureus Infections The present invention also encompasses methods of using the immunogenic compositions described herein. By way of example, one embodiment of the invention provides a method of inducing an immune response against S. aureus comprising administering to an individual an immunogenic amount of any of the immunogenic compositions described herein. One embodiment of the present invention provides a method of preventing an individual from contracting S. aureus, or a method of preventing S. aureus infection, or reducing the severity or delay of at least one symptom associated with an infection caused by S. aureus Methods of Attacking The methods comprise administering to the individual an immunogenic amount of any of the immunogenic compositions described herein. One embodiment of the present invention provides a method for treating or preventing a staphylococcal infection in a subject, a disease or condition associated with Staphylococcus, the method comprising administering to the individual a therapeutically or prophylactically effective amount The step of an immunogenic composition as described herein. In some embodiments, methods of treating or preventing a staphylococcal infection, disease, or condition include human, veterinary, animal or agricultural treatment. Another embodiment provides a method of treating or preventing a staphylococcal infection, a disease or condition associated with Staphylococcus in an individual, the method comprising producing a multi- or monoclonal antibody preparation from the immunogenic composition described herein and The antibody formulation is used to confer passive immunity to an individual. One embodiment of the invention provides a method of preventing staphylococcal infection in an individual undergoing a surgical procedure, the method comprising the step of administering to the individual a prophylactically effective amount of an immunogenic composition described herein prior to a surgical procedure. An "immune response" to an antigen or immunogenic composition is the subject's humoral and/or cell-mediated immune response to a molecule present in the relevant antigen or vaccine composition. For the purposes of the present invention, a "humoral immune response" is an antibody-mediated immune response and includes the induction and production of an antibody that recognizes an antigen in an immunogenic composition of the invention and binds thereto with a certain affinity, and "cell-mediated" The immune response is an immune response mediated by tau cells and/or other white blood cells. A "cell-mediated immune response" is initiated by the binding of an antigenic antigenic determinant to a class I or π major histocompatibility complex (MHC) molecule, CD1 or other non-canonical MHC-like molecule. Thus, antigen-specific CD4+ T helper cells or CD8+ cytotoxic T lymphocytes ("CTL") are activated. CTLs are specific for peptide antigens that bind to proteins encoded by classical or non-classical MHC and that appear on the cell surface. 149159.doc -56· 201110977 CTL helps to induce and promote intracellular destruction of intracellular microorganisms or to solubilize cells that infect these microorganisms. Another aspect of cellular immunity includes antigen-specific responses of helper T cells. The role of helper T cells is to assist in stimulating the function of non-specific effector cells and to concentrate their activity, and the cells to which these effector cells are directed may present peptides or other antigens on their surface that bind to classical or non-classical MHC molecules. "Cell-mediated immune response" also refers to the production of cytokines, chemokines, and other such molecules produced by activated T cells and/or other white blood cells, including those derived from CD4+ and CD8+ T-cells. The ability of a particular antigen or composition to stimulate a cell-mediated immune response can be determined by a number of assays, such as lymphocyte proliferation (lymphocyte activation) assays, CTL cytotoxic cell assays, by analysis of antigen specificity in sensitized individuals. T-lymphocytes, or by measuring cytokines produced by T cells in response to antigen re-stimulation. These analytical methods are well known in the art. See, for example, Erickson et al. (1993) ·/. 1 5 1:41 89- 4199, and Doe et al. (1994) V. J. 24: 2369-2376. As used herein, "treatment" (including variations thereof such as "treat" or "treated") means any one or more of the following: (1) prevention of infection or reinfection, such as traditional vaccines; Π) reducing the severity of symptoms or eliminating symptoms; and (iii) substantially or completely eliminating the pathogen or condition. Therefore, treatment can be carried out according to a preventive method (before infection) or a treatment method (after infection). In the present invention, prophylactic treatment is a preferred mode. According to a particular embodiment of the invention, compositions and methods are provided for treating (including prophylactic and/or therapeutic immunization) host animals against microbial infections (e.g., bacteria, such as staphylococcus). The methods of the invention are useful for conferring prophylactic 149159.doc -57-201110977 and/or therapeutic immunity. The method of the invention of the present invention can also be carried out for individuals who use the research application. The king's object, such as "mammals," means that humans or non-humans move you = animals refers to any animal classified as a mammal, including humans, brushed animals and farm animals, as well as zoo animals and pet companion animals. Such as 宕 0 0 Λ such as raising pets and other livestock, including

(but not limited to) cattle, sheep, cedar, pigs, horses, rabbits, goats U and the like. Better accompanied by animals and dogs. The mammal is preferably human. As used herein, the terms "chicken w θ immunogenic amount" and "immunologically effective amount" mean that the antigen or immunogenic composition is sufficient to elicit an immune response (cell (Τ cell) or body fluid (Β·cell or antibody) 4 of the reaction or both, as measured by standard analysis known to those skilled in the art. The amount of a particular combination in the composition is generally calculated based on the total polysaccharide (the bound polysaccharide of the combination and the unbound polysaccharide). For example, in a dose of 100 mcg CP5 polysaccharide, a cp5 conjugate containing 20% of free polyxon will have about rib meg binding to CP5 _ and about 20 mcg unbound cp5 multienzyme. When calculating the conjugate dose, it often does not consider the proportion of protein in the conjugate. The amount of conjugate can vary depending on the serotype of the staphylococcus. In general, each dose will comprise from 1 to 1 mc mcg polysaccharide, especially from 0.1 to meg polysaccharide, and more particularly to (7) meg polysaccharide. The "immunogenic amount" of the *multiple components in the immunogenic composition may be different, and each may contain 1 mcg, 2 mcg, 3 mcg, 4 meg ^ 5 meg &gt; 6 meg &gt; 7 meg &gt 8 mcg . 9 meg ^ 10 meg ^ 15 meg, 20 mcg, 30 mcg, 40 mcg, 5 〇 mcg, 6 〇 mcg, 7 〇 149159.doc • 58- 201110977 meg, 80 meg, 90 meg or about loo mcg Any specific polysaccharide antigen. Staphylococcus aureus "invasive disease" is the isolation of bacteria from a sterile site under normal conditions in which clinical signs/symptoms of the disease are present. Under normal circumstances, the body parts of the body include blood, CSF, pleural fluid ~ liquid, peritoneal fluid, joints / synovial fluid, bone, internal body parts (lymph nodes, brain, heart, liver, spleen, vitreous, f, sputum , ovary) or other sterile parts under normal conditions. Clinical conditions that characterize invasive diseases include bacteremia, pneumonia, cellulitis, osteomyelitis, endocarditis, septic shock, and other conditions. The effectiveness of the antigen as an immunogen can be determined by proliferation assays, by cell lysis (such as chromium release assays that measure the ability of T cells to lyse their specific target cells) or by measuring the extent of B cell activity (by amount) The amount of circulating antibodies specific for the antigen in the assay is measured. The immune response can also be measured by measuring the amount of the antigen-specific antibody induced by the antigen, and the sputum is enhanced by measuring the antibody thus induced to enhance the ability of the specific white blood to regulate the cells. Ability as described in this article. The immune response can be measured by the host immunized with the administered antigen. For example, 5, if the antigen to which the right immune response is directed is a bacterium, the degree of protection induced by the antigen in the immunogenicity is determined by the percentage of survival or the percentage of mortality after H is challenged by the fine g cell. Measurement. In a well-known example, the amount of protection can be measured by measuring at least one symptom associated with bacterial infection (e.g., fever associated with infection). The amount of each antigen in a multi-antigen or multi-component epidermal or immunogenic composition will vary with each other component and can be determined by methods known to those skilled in the art. Such methods 149159.doc -59· 201110977 include procedures for measuring immunogenicity and/or in vivo efficacy. In some embodiments, the term "about" means 20 〇 /. Within, preferably within 1%, and more preferably within 5%. The invention further provides antibodies and antibody compositions that specifically and selectively bind to the serotype 5 or 8 capsular polysaccharide or immunogenic conjugate of the invention. In some embodiments, the antibody is produced following administration of a serotype 5 or 8 capsular polysaccharide or immunogenic conjugate of the invention to an individual. In some embodiments, the invention provides purification and isolation of antibodies against one or more serotype 5 or 8 capsular polysaccharides or immunogenic conjugates of the invention. In some embodiments, the antibodies of the invention are functional, as measured in an animal potency model or via a conditioned swallow cell killing assay, based on killing bacteria. In some embodiments, an antibody of the invention confers passive immunity to an individual. The invention further provides a polynuclear (tetra) molecule encoding an antibody or antibody fragment of the invention, and a cell or cell strain producing the antibody or antibody composition of the invention (such as for recombinant production of antibodies) using techniques well known to those skilled in the art. A fusion of tumor cells or other engineered cell lines) or a transgenic animal. The antibody or antibody composition of the present invention can be used in a method for treating or preventing a staphylococcal infection, a disease or a condition associated with a Staphylococcus aureus in an individual, the method comprising producing a multi-plant or monoclonal antibody preparation and using the antibody or antibody The composition imparts passive immunity to the individual. The antibodies of the invention may also be used in diagnostic methods, such as the presence or quantification of the presence of CP5, CP8 or combinations thereof. Several animal models known in the art can be used to assess the efficacy of any of the immunogenic compositions described herein. For example: 149159.doc 201110977 Passive mouse sepsis model: Mice were passively immunized with immunized IgG or monoclonal antibody intraperitoneally (I.p_). After 24 hours, the mice were challenged with lethal S. aureus. Intravenous (i.v. or i.p.) administration of bacterial challenge ensures that any survival rate can be attributed to the specific in vivo interaction of the antibody with the bacteria. The bacterial challenge dose was determined to be the dose required to achieve approximately 2% of the unimmunized control mice to achieve lethal sepsis. The survival rate study can be statistically assessed by Kaplan-Meier analysis. Active immunization and challenge models: In this model, mice were actively immunized with the target antigen subcutaneously (SC) at weeks, 3, and 6 weeks (or similar time courses known to those skilled in the art), and in the first Attacks were performed by intravenous or intraperitoneal route for 8 weeks (or other similar time courses known to those skilled in the art). Calibration The bacterial challenge dose achieved a survival rate of approximately 2% in the control group over a 14 day period. The survival rate study can be statistically assessed by Kaplan-Meier analysis. Passive Infective Endocarditis Model: A passive immunization model of infective endocarditis (IE) previously used with S. aureus has shown that clfA can induce protective immunity. See Vernachio et al. (2〇〇6) ά C/iemo. 50:511-518. In this IE model, rabbits or rats were used to simulate clinical infections including central venous catheters, bacteremia, and hematogenous spread to peripheral organs. A single intravenous injection of a single or multiple antibodies specific for the target antigen is administered to rabbits or rats implanted with a sterile aorta. After 24 hours, the animals were challenged intravenously with a heterologous staphylococci strain or a mrsa strain. Next, after 48 hours of challenge, the heart, kidneys, and blood were collected and maintained. Then measure the heart valve, kidney and blood. 149159.doc -61 - 201110977 The frequency of staphylococcal infection in the fluid. In one study, when using mrse ATCC 35984 or MRSA PFESA0003 to challenge animals, use

A multi-drug antibody preparation or a monoclonal antibody of ClfA showed a marked decrease in the infection rate. See Vernachio et al., supra. Passive Infective Endocarditis Model: This infective endocarditis model is also suitable for active immunological studies. Rabbits or rats were immunized intramuscularly (im) with the target antigen and challenged with S. aureus via the intravenous route two weeks later. Pyelonephritis model: In the pyelonephritis model, mice were immunized with the target antigen at weeks, 3, and 6 weeks (similar time periods known to those skilled in the art). At week 8, the animals were challenged by, for example, intraperitoneal injection of, for example, 17 x 8 cfu of S. aureus PFESA0266. After 48 hours, the kidneys and/or other tissues are collected and cultured. Finally, the community forming units of the challenged bacteria in the kidney and/or other tissues are counted. This model assesses systemic spread in animals. Monitoring functional antibodies using opsonophagocytic killing assays This assay uses differentiated effector cells from cells isolated from donor human blood using LYMPHOLYTE®-poly solution (Cedarlane laboratories limited, Ontario, Canada) according to the manufacturer's protocol. Strain (eg HL60) or polymorphonuclear cell (PMN). The effector cells were resuspended in assay buffer (modified Eagle's medium containing 1% bovine serum albumin) at a concentration of about 2X 1 〇 7 cells/ml and placed at 37. (: in the incubator until ready for use. S. aureus strain PFESA0266 was grown overnight on tryptic soy agar plates. Bleed the bacterial cells, washed twice and resuspended in 5% glycerol in assay buffer until 〇d600 = 1, this value is equal to approximately 5X1 〇8 149159.doc • 62- 201110977 cfu/ml concentration. 1 aliquot of frozen bacterial suspension and stored at -40 C until ready for use. The solution was thawed and adjusted to a concentration of 1 〇 6 Cfu/mi in assay buffer and placed on ice. The assay was performed using a sterile 96 deep well 1 mi polypropylene plate. Prepare twice the serial dilution of the antibody sample (50 Μΐ), then add 3 〇〇 analysis buffer to the antibody mixture. Add bacteria to the assay plate (5 〇 and place on the spin shake for 30 minutes at 4 。. Perform the conditioning step, then add 5 〇 μ1 Human complement (final; 1%). Finally, 5 〇y effector cells (concentration of 1 〇 7 cells/ml) were added to the assay plate, and the suspension was thoroughly mixed by repeated pipetting. 5〇μΐ aliquot of the suspension 1 〇 serial dilution in sterile 1% saponin solution, vortexing to minimize bacterial agglutination, and two strokes were applied to chymotrypsin soy agar. Under continuous mixing using a rotary barbecue shaker, at 37 The assay plate was incubated for 1 hour. At the end of the incubation, the 50 μΐ monolith of the core float was serially diluted 1 〇 in a sterile i % saponin solution and mixed by vortexing to minimize bacterial agglutination. And applied to trypsin soy agar in duplicate. The number of viable efus in wells with bacteria, antibodies, complement and effector cells was determined at 6 min, 〃 lacking antibodies but containing bacteria, complement and effector cells The percentage of surviving cfu is used to calculate the percentage of kill. A control containing bacteria, complement and serum is included to regulate any reduction in Cfu due to agglutination. Complement adsorption for humans adsorbed by S. aureus strains PFESA〇266, p PFESAG27G Serum was used as a source of complement in the analysis. Golden yellow grape (4) strains grew overnight on 3rcMTSA plates. 149159.doc -63 - 201110977 The plate was scraped off and resuspended in sterile PBS. Bacterial cells were centrifuged at 10,000 rpm for 10 minutes at 4 rpm and the cell pellet was resuspended in human serum for adsorption. Serum was incubated with bacteria at 4. Incubate for 30 minutes on a rotary nutrator. Centrifuge the cells, transfer the serum to another tube containing the bacteria, and repeat the adsorption step for another 30 minutes. Finally, centrifuge the cells and pass the serum through the 〇.2 A micron filter followed by a 0.5 ml aliquot in liquid nitrogen. Method II-0PA ' uses HL-60 cells to differentiate HL-60 cells according to S. Romero-Steiner et al. 'Clin Diagn Lab Immun.1 4(4) (1997), pp. 415-422. The collected HL-60 cells were resuspended in assay buffer (modified Eagle's medium containing 1% bovine serum albumin) at about 1 8 cells/ml and placed at 37. 〇 Incubate the box until ready for use. S. aureus strains were grown overnight on tryptic soy agar plates. The bacterial cells were scraped off, washed twice and resuspended in assay buffer containing 5% glycerol until 〇D6Qg=1, which is equal to about 5χΐ〇8 cfu/m of frozen bacterial suspension! An aliquot of m丨 was stored and stored under _4 直至 until it was ready for use. The frozen bacterial suspension was thawed and adjusted to a concentration of 1 〇 6 cfu/m b in assay buffer and placed on ice. The assay was performed using a sterile % deep well 1 m] polypropylene plate. Two-fold serial dilutions (25 μΐ) of the individual antibody samples were prepared, followed by the addition of 15 μy analytical buffer to the antibody suspension. Bacteria (25 μl) were added to the assay plate and placed on a rotary shaker for 30 minutes, followed by the addition of 25 μm human complement (final concentration 〖%). Finally, 25 μl of HL_6 〇 cells (1〇7 cells/ml) were added to the assay plate, and the suspension was thoroughly mixed by repeated pipetting. 25 W aliquot of the suspension 149I59.doc •64· 201110977 The sputum is fed into the sterile 1 〇/0 saponin solution. The β is continuously diluted and vortexed to form bacteria. The agglutination was minimized, and the two were applied to the trypsin and the two were applied. Use the Rotary Grill 牟 37. Wide w (10) wood-type oscillators were continuously mixed under the 1982 &quot; analysis board for 1 hour. At the end of the incubation, the suspension, etc., were sampled in a ML%-free solution, mixed in a continuous dilution, mixed by vortexing and applied in duplicate to chymotrypsin soy agar. Determined at 60 minutes. The ratio of the number of surviving cfus in the wells with bacteria, antibody complement and HL-60 cells was compared with the number of surviving cfus in the tubes lacking the antibody to 3 with fine halo, complement and HL-60. This includes the inclusion of bacteria, complement, and mAb, ▲ „ mt mt &amp; α to adjust for any cfu reduction due to agglutination. The following examples are provided by way of illustration and not limitation. EXAMPLES Example 1: Preparation of S. aureus serotype 8 capsular polysaccharide In this example, S. aureus serotype 8 serotypes of various molecular size ranges were described. The structure of the S. aureus serotype 8 capsular polysaccharide repeating unit is shown in Figure 。. The methods described herein are effective in producing serotype 8 capsular polysaccharides in the range of about 20 kDa to 700 kDa in knives. High molecular weight serotype 8 capsular polysaccharide having a molecular weight in the range of 5 〇 kDa to 700 kDa can be isolated and purified by appropriately selecting conditions. For use in immunogenic compositions, serotype 8 capsular polysaccharides having molecular weights ranging from 7 〇 kDa to 3 〇〇 kDa and many desired ranges can be isolated and purified. Based on the growth characteristics and the amount of capsular produced, the strain PFESA0005 or sputum was used to produce serotype 8 capsular polysaccharide. The capsular membrane was shown to be the same from the strain PFESA0005 or PFESA0286, H9159.doc •65·201110977. A serotype 8 capsular polysaccharide is produced, and the strain is grown in a complex medium mainly composed of a carbon source (lactose or sucrose), hydrolyzed soybean powder as a nitrogen source, and a trace metal. The strain is grown in a bioreactor for 2 to 5 days. The sample was removed in the heat c treatment to test the content of staphylococcal enterotoxin B (SEB) in the culture. The concentration of SEB in the acid yeast was (10) ng/ml in the presence of 0.05% polysorbate 80. Previous experiments have shown that the culture is hot pressed! The hourly reduction of SEB content is less than 〇1 ng/mi, which is below the detection limit of the TECRA kit. - The diafiltration-filtered ethanol (4) was loaded on a Q·Sepharose column and dissolved by a heart (four) gradient as described above. The fractions were analyzed by hydrazine thiol analysis and two-way immunodiffusion test (testing for the presence of serotype 5 polysaccharide) and acid salt analysis (analysis of the presence of teichoic acid (TA)). The presence of serotype 8 polysaccharide was detected in fractions 35 to 95 (Fig. 2 to 4). To reduce the contamination of the teichoic acid, the fractions 35 to 75 were pooled and any residual teichoic acid was oxidized with sodium metaperiodate to remove it by diafiltration against 3K of re-distilled water. The serotype 8 capsular polysaccharide used to prepare the conjugate is purified by two different methods that affect the release of the capsule from the cell and reduce the molecular weight of the polysaccharide depending on the temperature and the low pH. The molecular weight obtained depends on the time, temperature and value of the hydrolysis step. Serotype 8 capsular polysaccharides were characterized using the techniques described in Table 1. 149159.doc -66 - 201110977 Table 1: Characterization of purified S. aureus serotype 8 capsular polysaccharide

Specificity analysis of residual protein Lorry colorimetric assay Residual nucleic acid 260 nm scanning residual phosphonium acid phosphate colorimetric analysis residual peptidoglycan HPAEC-PAD size SEC-MALLS Composition HPAEC-PAD identity 1H-NMR or Specific mAb reaction 0-acetamidine 1H-NMR concentration MALLS-RI or HPAEC-PAD The capsular polysaccharide produced by the following method can be fully characterized by low content of protein, nucleic acid, peptidoglycan and teichoic acid contaminants. Pure polysaccharide. In the first method, after the capsular polysaccharide is released from the cells and the molecular weight is lowered, the preparation is treated with an enzyme mixture (e.g., ribonuclease, deoxyribonuclease, lysozyme, and protease) to digest impurities. After incubation, residual impurities were precipitated by the addition of ethanol (final concentration of about 25%). After removing residual ethanol, the capsular polysaccharide-containing solution was loaded on an anion exchange column (Q-Sepharose) and dissolved with a linear salt gradient. The fractions containing the capsular polysaccharide were pooled and treated with sodium molybdate. This treatment resulted in residual linal acid contaminants being oxidatively hydrolyzed but not affecting serotype 8 capsular polysaccharides. The reaction was quenched by the addition of ethylene glycol. The material was concentrated and dialyzed against distilled water to remove any residual reagents and by-products. The second method is used to produce capsular polysaccharide without the use of enzymes to digest various cell-derived impurities. In this method, the capsular polysaccharide is released from the cells and the molecular weight of 149159.doc-67-201110977 is lowered, and the hydrolyzed fermentation broth is clarified by microfiltration, followed by ultrafiltration and diafiltration. The solution is treated with activated carbon to remove impurities. After carbon treatment, the material was treated with sodium metaperiodate to oxidize the residual teichoic acid, followed by quenching with propylene glycol. The material was concentrated and dialyzed against distilled water to remove any residual reagents and by-products. The preparation of the preparation using either method yields a pure capsular polysaccharide having a low content of protein, nucleic acid and teichoic acid contaminants. The method is used to produce a desired high molecular weight polysaccharide in a particular range by varying the hydrolysis conditions τ. Examples of capsular polysaccharides obtainable by the methods described herein are shown in Table 2 below. Each batch of purified blood/moon type 8 capsular polysaccharide has high purity as indicated by non-phospholiic acid (TA), peptidoglycan, and low residual protein. See Table 2. The low molecular weight range is from 20.4 kDa to 65.1 kDa, and the purified polysaccharide is subjected to high enthalpy (about 100%). The degree of nucleic acid contamination is low (012_2 Μ%). Table 2: Characterization of serotype 8 capsular polysaccharide preparation 1 2 3 (kDa) (g/mol) (Lori) % (w/w) (260 nm scan) % (w/w) NMI % 310 27.0 ! 2 438 29.0 2 4 179 ^ 0-37 (10) 1 分子量 膜 膜 之 之 分子量 分子量 分子量 分子量 分子量 分子量 分子量 分子量 分子量 分子量 分子量 分子量 分子量 分子量 分子量 分子量 分子量 分子量 分子量 分子量 分子量 分子量 分子量 分子量 分子量 分子量 分子量 分子量 分子量 分子量First, it is purified by fine 1: large and large listening 'subsequent selection of the desired molecular weight range 1 and the pH of the transfer step and heating conditions. The heat treatment of the Staphylococcus aureus (4) culture solution is a treatment step between the fermentation and the recovery of 0.94 2 0.12 10C 10C 108 H9159.doc -68· 201110977 vinegar recovery. This treatment step uses heat to treat the pH-adjusted culture solution for a specific period of time. The goal of heat treatment at low pH is to kill and die. Tianjian inactivates the enterotoxin, releases the cell-bound polysaccharide and reduces the molecular weight to the desired size. In these targets, 'the processing time required for this step' is the slowest to lower the molecular weight. Therefore, other goals must be achieved within the processing time considered. Heat Treatment: Determine the pH and degree conditions used to select various molecular weight ranges for the capsule ride. These studies used a 15 l Biolafme Fementei. Transfer the culture medium to the acid fertilizer by using snail (4). The pH of the culture medium was adjusted with concentrated sulfuric acid using a search speed of about 200 rpm. Then, the temperature of the culture solution is raised to a set value. The heat treatment timing is started as soon as the temperature reaches the set point. When the desired treatment time is reached, the sigma solution is cooled to the 'cover. The samples in the treatment were obtained, and the polysaccharide concentration and molecular weight were determined by HPLC and SEC-MALLS systems, respectively. Molecular weight (MW) data were used for kinetic analysis. The MW distribution was measured over time at ρ Η 3 5, 4 〇 and 5 。. See Figure 3 a. Kinetic analysis of the weak acid hydrolysis of the polysaccharide was carried out using purified serotype 8 capsules obtained from this treatment. The purified poly(tetra) solution was adjusted to the desired pH with sulfuric acid for experimental use. Transfer 5 mL of the solution to each of the 15 'centrifuge tubes. Place the tube in an oil bath equipped with a precision temperature control system. Centrifuges and tubes were taken every predetermined time and quenched in an ice bucket. At the end of the experiment, an aliquot of the i M Tris buffer (pH 7.5) was added to the sample to adjust the positive value back to about 7. Samples were analyzed by the SEC_MALLS system. Na data used (four) mechanical analysis. Under ΡΗ 3·5, the temperature is determined by the time of day (10). 149159.doc -69- 201110977 The influence of cloth. See Figure 3B. As shown in Figure 3A, a lower pH is more effective in reducing the molecular weight of the polysaccharide. pH 5, 95 can be used. (:, 15 minutes to 120 minutes to produce a molecular weight of 300 kDa to 600 kDa. Similarly, pH 4, 95 can be used. 匸, 15 minutes to 120 minutes to produce a molecular weight of 250 kDa to 450 kDa. pH 3.5 '95. (:, for 15 minutes to 120 minutes to produce a molecular weight of 120 kDa to 450 kDa. As shown in Figure 3B, the higher the temperature, the faster the hydrolysis rate and the resulting polysaccharide The broader the molecular weight range, the lower molecular weight range of 55 C (relative to 95%) is produced at the same pH value. In addition, Figure 4 shows the molecular weight of the purified CP8 and the weak acid (pH 3 5, 95. Correlation of hydrolysis treatment time. Purified polysaccharide is the final product obtained from the recovery treatment detailed above. As shown in Fig. 4, prolonging the heat treatment time of S. aureus PFESA0005 strain under Yang 3 5 can produce molecular weight. Small CP8, while shortening the heat treatment time at pH 35 can produce CP8 with higher molecular weight. Depending on the length of heat treatment at pH 3.5, the molecular size of serotype 8 capsules is about 8〇kDa to about 22 〇kDa fan As shown in Figure 4, the correlation between the heat treatment time at low pH and the molecular size of purified cp8 can be used to assess the processing time required to produce purified cholestases with specific molecular weight ranges. Thus, as indicated above, the entire molecular weight range of 2 kDa to more than 5 〇〇 kDa can be produced, released and purified. Therefore, these methods can be used to produce a capsule having a specific desired molecular weight of 149159.doc 201110977. The sugars are shown in Table 3. The molecular weight range produced is relative to the sucrose (where the peak molecular weight is in the range of 87 〇 3 to i 〇 8), indicating that sufficient is obtained by the methods described herein. Characterized molecular weight range. Particularly advantageous polysaccharides in the high molecular weight range (in the range of 7〇^^&amp; to 3〇〇kDa or 7〇kDaJ* 150 kDa) can be obtained by using capsular polysaccharides with carrier molecules or proteins, In combination, it was used to make an immunogenic composition (see Table 3). The conditions for producing a CP8 film polysaccharide having a molecular weight ranging from about 8 Å to 120 kDa were as follows: 95 C 'pH 3.5, which lasted 3 minutes. 3 produces specific High molecular weight range of Jinqing type 8 capsular polysaccharide

100 -~~~~~ _87___ Example 2 ·Serotype 8 capsule combined with polysaccharide CRM197] The method and characterization analysis for the production of S. aureus serotype 8 capsular polysaccharide. CRMl97 conjugate. Different binding chemistries have been developed for the binding of S. aureus serotype 8 capsular polysaccharide to this carrier protein. For example, the use of PDPH (3-(2_t-definyldithio).propanol I49159.doc •71 - 201110977 肼) binding can produce a covalent thioether bond between cp and a carrier protein. Alternatively, a single carbon or zero carbon linker can be produced between the CP and the carrier protein using a combination of CDI/CDT (1,1-mono-2-n-mial/1, !-yl-di-oxazole). Serotype 8 capsular polysaccharide was combined with CRM... by PDPH binding chemistry. The PDPH binding chemistry is a multi-step process that involves activating the polysaccharide, removing the thiol protecting group, purifying the activated polysaccharide intermediate, activating and purifying the CRM, 9? protein, and binding the activating component, followed by purification. After introducing a thiol group-containing linker into the polysaccharide and introducing a haloacetic acid group into the CRMl97 protein carrier, the S. aureus serotype 8 capsular polysaccharide is linked to the protein carrier via a thioether bond. The bromoethenyl group is introduced into the CRM 9-9 protein by the reaction of an amine group with N-hydroxybutylidene bromoacetate. In order to produce a thiolated polysaccharide, the carbodiimide-activated formate group of N-ethyl decylamino mannitol in the polysaccharide is coupled to a thiol reactive doped difunctional linker 3-(2- Amidino group of pyridyldithio)-propionyl (PDPH). The thiol of the PDPH thiolated polysaccharide purified by dtt reduction and purified by SEC on a Sephadex G25 column is reacted with the bromide group of the activated protein to form a bond between the polysaccharide and the carrier protein by bromine displacement. Covalent thioether bond. The unreacted bromoethane group is "capped" with cysteamine hydrochloride (2-aminoethyl thiolate). The reaction mixture was then concentrated and diafiltered. The remaining unbound bromoacetyl group is capped with cysteamine hydrochloride to ensure that no reactive oxime groups are present after binding. Thus, after bromine displacement, a covalent bond is formed between the thiol end of the cysteamine and the acetamidine group on the lysine residue. 1 - Thiolization of S. aureus serotype 8 capsular polysaccharide with PDPH: 149159.doc • 72· 201110977 The polysaccharide was first activated by thiolation of PDPH. The polysaccharide was mixed with freshly prepared PDPH stock solution (250 mg/mL in DMSO), EDAC stock solution (90 mg/mL in red distilled water) and MES buffer stock solution (0.5 Μ, pH 4.85). The final solution contained 0.1 M MES, and 2 and 4 mg/mL polysaccharide, while the serotype 8 capsular polysaccharide maintained a 1:0.6:1.25 polysaccharide:PDPH:EDAC weight ratio. Incubation was carried out for 1 hour at room temperature, followed by dialysis 4 times with respect to 1000 volumes of distilled water at 4 ° C to 8 ° C using a 3500 MWCO dialysis apparatus to remove unreacted PDPH. The PDPH-linked polysaccharide was produced with 0.2 Μ DTT and incubated for 3 hours at room temperature or overnight at 4 °C to 8 °C. Sephadex G25 resin and distilled water were used as the mobile phase, and excess DTT and reaction by-products were separated from the activated sugar by SEC. The thiol group of the dissolved fraction was analyzed by DTDP analysis, and the thiol-positive dissolved fraction dissolved in the vicinity of the void volume of the column was collected. The pooled fractions were analyzed by PAHBAH and 0-acetamidine analysis to determine the degree of activation, expressed as the percentage of moles of thiol-containing repeat units (molar concentration of thiol / molar concentration of repeating unit) ). The activated polysaccharide was lyophilized and stored at -25 °C until binding was required. The results of thiolation of the serotype 8 polysaccharide by PDPH are shown in Table 4. The serotype 8 polysaccharide has an activation degree in the range of 12% to 16%, corresponding to about 1 ligating molecule per 10 capsular polysaccharide repeating units to 1 linking molecule per 5 repeating units. 149159.doc -73- 201110977 Table 4: Activation of serotype 8 capsular polysaccharide by PDPH-reproducibility study serotype 8 polysaccharide-PDPH activation (% Msh/Mru) scale mg yield mg (〇/〇,w /w) 1 14 36 30(83) 2 16 30 27 (91) 4 16 38 42(110) 5 12 40 44(110) 2. Vector protein activation: The carrier protein is activated independently by bromination. CRM, 97 was diluted to 5 mg/mL with 10 mM phosphate buffered 0.9% NaCl (pH 7) (PBS), followed by a pH of 7.0 using a 1 Μ stock solution with 0.1 Μ NaHC03. N-hydroxybutylimine bromoacetate (BAANS) was added at a CRM197:BAANS ratio of 1:0.25 (w:w) using a BAANS stock solution of 20 mg/mL DMSO. The reaction mixture was incubated at 4 ° C to 8 ° C for 1 hour and then purified on Sephadex G-25 using SEC. Purified activated CRMm was analyzed by Lori analysis to determine protein concentration, followed by dilution to 5 mg/mL with PBS. Sucrose was added as a cryoprotectant up to 5% wt/vol, and the cold-beam activated protein was stored at -25 °C until binding was required. The bromoacetylation of CRM197 from the amino acid residue is extremely consistent, thereby activating 19 to 25 of the 39 amide acids available (see Table 5). This reaction produces activated proteins in high yield. 149159.doc -74- 201110977 Table 5: Yield and extent of CRM197 bromoacetylation Formulation 1 2 3 4 5 6 Scale Yield

23 38 35 35 35 48 85 87 77 94 87 104 Activated lysine (n=) 24 20 19 22 23 25 3 · Coupling reaction: Activated capsules and activated carrier proteins are prepared, even if one is combined Compound in the reaction. In the future, dry and thiolated, dissolved in 0.16 lanthanum borate (pH 8.95), mixed with thawed bromoacetylated CRMi97 and distilled water, so that the final solution contains 0 1 Μ borate, 1: 丨 weight ratio of CRMm : Polysaccharide and 1 mg/mL polysaccharide. Incubate at room temperature for 16 hours to 24 hours. Cysteine hydrochloride was added at a CRM of 97: cysteamine ratio of 1:2 (wt/wt) by using a mg/mL cysteamine stock solution dissolved in 〇1 M borate (pH 8 95). Salt was used to cap the unreacted ethidium bromide on the protein and incubated for 4 hours at room temperature. The capsular polysaccharide ^^^(1) conjugate conjugate was purified by 5 〇 diafiltration with respect to 0-9% NaCl using a 1 〇〇 κ polyether sulfone ultrafilter. The results of the research on the privatization of serotype 8 serotonin-main A-serum polysaccharide sulphur by PDPH showed that the activation degree of 'polysaccharide' was 12% to i 6%, Ming Yun Yun Gucheng - Wang 1 〇/〇干围, Corresponding to each of the polysaccharide repeating units, about 1 link is connected, and the first 5 molecules are connected to one repeating unit to connect one connecting molecule. The CDI/CDT binding chemistry method provides a one-step binding method for the C. capsular polysaccharide and the Crm197 junction 149159.doc -75- 201110977 CDI and CDT, wherein the polysaccharide is activated in an anhydrous environment (DMSO) to form a hydroxyl group with an available hydroxyl group. An imidazole or triazolyl phthalate moiety and a mercapto imidazole or decyl triazole moiety having a carboxylic acid. The addition of a protein carrier (contained in DMSO) causes nucleophilic displacement of the imidazole or triazole from the amine acid and formation of a urethane linkage (for the reactive hydroxyl group) and a guanamine linkage (for the active carboxylic acid). Both CDI and CDT binding chemistries produce serotype 8 capsular polysaccharide covalently linked to the carrier protein, according to the following instructions: the presence of sugars and proteins in the fractions of size exclusion chromatography, and the termination of glycolaldehyde Or amino acid analysis of a cysteamine hydrochloride-terminated conjugate. A summary of the results for preparing a plurality of batches of conjugates of capsular serotype 8 polysaccharides ranging from 20 kDa to 40 kDa (prepared by PDPH and CDI/CDT chemistry) is shown in Table 6 below. There was no significant difference in free capsular polysaccharide, poly-protein ratio and yield of the conjugates produced by the two binding methods. The antigenicity of the combined serotype 8 capsular polysaccharide is not altered by binding, as indicated by the identity precipitin line between the conjugate and the natural polysaccharide. Table 6: Characterization of the serotype 8 capsular polysaccharide crm197 molecular size prepared by two binding chemistry methods (MW or CP yield, chemical free sugar free egg modified chemical export ratio Kd (% &lt; 0.3), polysaccharide / Egg (%) Yield (%) (%) White matter (%) Offic acid white matter) CDI/CDT 46-62 54-55 0.8-0.9 22-25 &lt; 1 7-8 34/57 to 60/57 PDPH 34-70 61-83 0.6-0.9 15-41 ND 11-16 74-92% 149159.doc -76- 201110977 As indicated above, the methods described herein can be used to produce capsules with specific high molecular weight ranges Polysaccharide. A conjugate is prepared from a serotype 8 capsular polysaccharide having a pre-selected high molecular weight range that can be filtered and purified for use in an immunogenic composition. Table 7 summarizes the analysis of serotype 8 capsular polysaccharide conjugates wherein the serotype 8 capsular polysaccharide has a molecular weight in the range of from about 80 kDa to 120 kDa and utilizes imidazole binding chemistry. The molecular weight of the resulting conjugate is in the range of 595 kDa to 1708 kDa. The number of bound deaminic acids in each CRM197 ranged from a high value of 9 to a low value of 3. The free capsular polysaccharide ranges from a high value of 6% to a low value of 2%. Table 7: Conjugates of serotype 8 capsular polysaccharides with pre-selected molecular weight ranges. Test polysaccharide MW (kDa) yield (%) free sugar (%) MW, modified lysine by SEC-MALLS (kDa) 1 99 88 6 943 4 2 113 73 5 841 3 3 105 79 3 719 7 4 100 86 2 630 9 5 87 90 3 595 6 Both binding chemistries produce serotype 8 capsular polysaccharide covalently linked to the carrier protein . There was no significant difference in free capsular polysaccharide, serotype 8 capsular polysaccharide: protein ratio and the yield of the conjugate produced by the two methods. Example 3: - Step method (〇1^卩〇〇 compared to the complex €01/€01' method as described above, the method for making the immunogenic conjugate of the invention comprises using a binding chemistry method to make the capsule The polysaccharide is covalently bound to the carrier protein, and the binding chemistry includes CDI (1,1-carbonyldiimidazole), CDT (1,1-carbonyl-di-149159.doc •ΊΊ · 201110977 1'2'4-diode or 1) 1 &gt; 11 (3_(2_0 than dimethyldithio) _ propylene). Using CDI/CDT to produce a single carbon or zero carbon linker between the membrane polysaccharide and the carrier protein f, while using PDPH A covalent thioether bond is produced between the capsular polysaccharide and the carrier protein. The PDPH-based method is a multi-step method, including activation, removal of the thiol protecting group on the multi-enzyme, purification of the activated polysaccharide intermediate, activation And purifying the protein carrier, and binding the activating component, in which the S. aureus serotype 8 capsular polysaccharide is reacted with pDpH and carbodiimide in an aqueous solution such as 0.1 M MES to produce PDPH. a linked polysaccharide. The PDPH-linked polysaccharide reacts with a reducing agent to produce an activated polysaccharide. Purification. The carrier protein is reacted with N-hydroxybutyrimidine bromoacetate in aqueous solution to produce an activated carrier protein, which is then purified. The purified activated serotype 8 polysaccharide is then reacted with the purified activated carrier protein to produce serotype 8 Polysaccharide: carrier protein conjugate. In contrast, 'CDI-based and CDT-based methods are one-step or two-step binding methods' in which the capsular polysaccharide is activated in an anhydrous environment (ie, DMSO) to form an imidazole or three with available hydroxyl groups. An oxazolidine phthalate moiety and a mercapto imidazole or decyl triazole moiety having a slow acid. Addition of a protein carrier (containing in DMS oxime) causes nucleophilic displacement of the imidazole or triazole with an amine acid and formation of an amine phthalate Keys (for active hydroxyl groups) and guanamine bonds (for active carboxylic acids). Therefore, two methods based on CDI or CDT have been developed: a more complicated method and a simpler one-step method. In the more complicated method ten, golden yellow Staphylococcal serotype 8 capsular polysaccharide is combined with imidazole or triazole, lyophilized, and then reacted with CDI or CDT in an organic solvent such as DMSO to produce an activated serotype 8 148159.doc •78· 201110977 Activated serotype 8 polysaccharide, followed by reaction with carrier protein in an organic solvent to produce serotype 8 polysaccharide-carrier protein conjugate. One-step method is similar to complex method except for activated serum Type 8 polysaccharide was not purified before reaction with carrier protein. CDI/CDT complex method activated polysaccharide: serotype 8 polysaccharide mixed with 10 g triazole/g serotype 8 and; Donggan. The obtained cake was 2.0 mg serotype. 8 polysaccharide/ml was dissolved in DMSO. The water content was determined. A freshly prepared CDT stock solution (100 mg/mL in DMSO) was added until the molar amount of CDT was equal to water. Alternatively, the amount of CDT added can be adjusted to achieve a higher or lower degree of activation. Hold at 23 ° C for 30 minutes. Purification of activated serotype 8 polysaccharide: A solution of activated serotype 8 (ACP8) was poured into 25 volumes of water to remove excess CDT. It was concentrated to about its original volume on a 10 kDa PES film at about 1 mg/cm2 and diafiltered against at least 10 volumes of water. This step is completed in less than 4 hours. The diafiltered material was mixed with 10 g of triazole/g raw serotype 8 polysaccharide and lyophilized. Preparation of lyophilized CRM: CRM was dialyzed against a 10 kDa PES membrane in a constant volume relative to at least 10 volumes of 0.4% NaCl/5% sucrose. The protein concentration was measured, and sufficient diafiltration buffer was added to bring the protein concentration to 5.0 g/L, thereby obtaining a NaCl/CRM weight ratio = 0.8. Freeze dry CRM. Binding: The activated, diafiltered serotype 8 polysaccharide was dissolved in DMSO at 1 mg/mL. Add 100 mM borate solution until 2% v/v is reached. The CRM was resuspended at 2 mg/mL and, when completely dissolved, was combined with the ACP8 solution 149159.doc •79-201110977. Allow to react at 23 ° C for 20 hours. The conjugate reaction solution was poured into 24 volumes of 5 mM borate (pH 9.0) and stirred at room temperature for 1 hour. It was then adjusted to pH 7.5 with 0.5 Μ phosphate buffer (pH 6.5). It was filtered through a 5 micron filter and concentrated to the original volume on a 300 kDa PES film at a load of about 1 mg/cm2 and diafiltered against at least 10 volumes of water. The resulting concentrate was filtered through a 0.22 micron filter and stored at 2 °C to 8 °C. CDI/CDT-step method CRM丨97 matrix exchange: diafiltration CRM197 to exchange about 10 mM acidate / 80 mM NaCl / 5% sucrose whole matrix (pH 7) into 5 mM flavor 0 sit / 0 · 72% NaCl/15 m octyl-β-D-saccharide (pH 7). Exchange can remove phosphate and sucrose that are not conducive to bonding and will limit the amount of vaporized sodium delivered to the bond. The octyl-?-D-glucopyranoside is added to prevent the formation of particles after sterile filtration. Exchange of CRM197 matrix by tangential flow transition with respect to 5 mMem sit/〇.72%/15 mM octyl-Β-D-glucopyranoside (pH 7) (using 1 OK MWCO PES film The retention concentration is about 4 mg/mL, filtered by 1 diafiltration volume multiple). The typical membrane challenge is 2 g/ft2' and the final target Crm197 concentration in the matrix is 6 mg/mL. CRMi97 is stored at 2 ° C to 8 ° C. Activation/Binding·Staphylococcus aureus serotype 8 membrane vinegar activation/binding method consists of the following steps: 1) matrix exchange of CRM丨97; 2) combination of polysaccharides; 3) shell cold and freeze-dried CRMw And compounded polysaccharides; 4) solubilization; Dongganzhiduo and CRM〗; 5) activated polysaccharides; 6) activated polysaccharides combined with CRm197 149159.doc -80- 201110977; and 7) purified conjugates (dilution, Dialysis filtration, sterile filtration) Polysaccharide is combined with 10 g of 1,2,4-triazole excipient/g of polysaccharide. The excipient is added to the polysaccharide in powder form, and the solution is obtained after mixing at ambient temperature for less than 15 minutes. The combined polysaccharide and CRM197 were shell-frozen using a -75 ° C ethanol bath, respectively, and the volume per 1 L of the bottle was about 500 mL. In order to dissolve the polysaccharide, DMSO was added to the individual freeze-dried bottle containing the polysaccharide to obtain a suspension. Then transferred to an activation/binding reaction vessel for heating. DMSO was added until a concentration of 2 g/L was obtained. A clear solution was obtained when the suspension reached about 45 ° C under mixing. The solution was then cooled to 23 ° C ± 2〇C. In order to dissolve crm197, to contain CRM197 DMSO was added to individual lyophilized vials to obtain a suspension, which was then transferred to a second vessel for mixing. DMSO was added until a concentration of 2 g/L was obtained. A clear solution was usually obtained in less than 15 minutes. Sampling the polysaccharide/DMSO solution Karl Fischer analysis to determine the moisture content. CDT was prepared in 100 mg/mL solution in DMSO and added based on the measured moisture content. At 23 ° C ± 2 ° C, under mixing The CDT solution was added continuously for about 5 minutes. The reaction was carried out at 23 ° C ± 2 ° C for a minimum of 30 minutes. The reactants were sampled to determine the degree of activation (UV 220 / 205 nm) 'Next 100 mM sodium borate (pH 9) was added A 1.5% aqueous solution was obtained. Then, the reaction solution was stirred at 23 ° C ± 2 ° C for a minimum of 30 minutes. In order to bind the activated polysaccharide to CRM197, DMSO was added to obtain a reaction concentration of 0.8 mg / mL. CRM197 dissolved in DMSO was followed by Add 149159.doc -81- 201110977 to the activated polysaccharide solution under mixing. The reactants are at 23 (: ± 2) (: stirring for a minimum of 4 hours. Under mixing, the reaction solution is added to 5 mM tetraboric acid by mixing Dilute H) times in sodium (pH 9) Activity (4). The solution of (4) is passed through a 5 (four) transition device and concentrated to a target retention concentration of 2 g/L. For use, the regenerated cellulose membrane is filtered through a volume of 20 diafiltration filters to 5 succinate (pH 7). Tangential flow filtration. Typical membrane loading was g/ft2. The purified conjugate was passed through a 0.22 micron filter' and stored at 2 〇c to 81. Example 4: Binding of serotype 8 capsular polysaccharide using a one-step method and a complex binding method This example shows that Weidu (4) with a preselected molecular weight range can be used for binding in a one-step or complex method. First, a larger polysaccharide is produced from the fine cells, and the molecular weight range obtained by purification can be controlled by the pH of the hydrolysis process of the example and heating (as shown in Table 3). In this example, the serotype 8 pods are selected. 8 batches of membrane polysaccharides with molecular weights ranging from about 8 〇kDa to about kDa, and using activation (for serotype 8 laughter, 1,1-Mec-di-(1,2,4-triazole) The binding is carried out. See Table 8. The molecular weight of the resulting conjugate is in the range of 595 〇 &amp; to 17 〇 8 kDa. The number of bound lysines in each CRM is in the range of high 13 to low 3. Between the high value of 11% and the low value of 1%. 149159.doc •82· 201110977 Table 8: Serotype 8 capsular polysaccharide conjugate method prepared with 80 kDa to 120 kDa capsular polysaccharide Method for the determination of polysaccharide MW(kDa) sugar Yield (%) free sugar (%) MW, one step by SEC-MALLS (kDa) lysine 1 98 86 1 751 11 2 89 80 1 675 13 3 108 76 4 1073 5.0 4 108 69 4 819 5.2 5 89 85.1 8 1708 10 6 100 94.0 11 1577 5 Complex method 1 99 88 6 943 4 2 113 73 5 841 3 3 105 79 3 719 7 4 100 86 2 630 9 5 87 90 3 595 6 Example 5: Evaluation of the primary serotype 8 capsular polysaccharide combined with the alkali treated jk clear type 8 capsular polysaccharide in a murine bacteremia model. Evaluation of the presence of pre-existing serotype 8 capsular polysaccharide for induction function The importance of the 0-acetyl group of the serotonin response to the capsular polysaccharide conjugate. The serotype 8 capsular polysaccharide was removed under weak alkaline conditions, and both NMR and ion chromatography (1C) confirmed There is no 0-acetamidine in jk clear type 8 film polysaccharide de-0-Ac-CRM. The combination of de-O-Ac CP8 polysaccharide and CRM by PDPH chemistry to prepare CP8 de-O-Ac-CRM The substance, as described in Example 2. The serotype 8 capsular polysaccharide conjugate unexpectedly showed that the ethyl thiol group was not detected by the 1C method. This can be attributed to the structure, the oxime-acetylation site and other gold. The S. aureus capsular polysaccharide is different, which in turn can remove or modify the acetamyl group in the serotype 8 capsular polysaccharide of 149159.doc -83- 201110977 during the binding. The murine bacteremia model is used to evaluate the native serotype 8 The effect of capsular polysaccharide on the binding of alkali-treated serotype 8 capsular polysaccharide to CRM197. Female BALB/c mice in each group (15/ ) Were seeded in the first six weeks and 0,3 1 meg serotype 8 capsular polysaccharides de-0-Ac-CRM or 1 pg serotype 8 capsular polysaccharide O-Ac-CRM. The vaccine was formulated with 22 meg AlP〇4. Animals were challenged with S. aureus PFESA0003 and bacteria in the blood were counted after 3 hours. The data showed that the reconstituted bacterial efu in the blood of the animals immunized with the untreated native serotype 8 capsular polysaccharide conjugate was statistically significant (ρ = 0362), as determined by Student's t-test (Table 9). In the animals immunized with the alkali-treated serotype 8 capsular polysaccharide conjugate, the reconstituted bacterial efu in the blood was similar to the physiological saline control group. Table 9: Serotype 8 capsular polysaccharide-CRM197 conjugate reduces bacteremia caused by S. aureus PFESA0003 in mice Antigen strain/dose LogCFU/blood (P value) Physiological saline 4.35 PFESA0003 CP8 de-0 -Ac-CRM „ 4.45 1.14x10s CP8 O-Ac-CRM 3.93 0.03 Example 6: Evaluation of primary serotype 8 capsular polysaccharide combined with alkali-treated serotype 8 capsular polysaccharide evaluation serum in a murine bacteremia model The ability of the type 8 capsular polysaccharide conjugate to protect mice in the pyelonephritis model showed a significant decrease in the bacterial count in the blood of mice infected with S. aureus in the peritoneum compared to the control group immunized with PBS. In the study, the efficacy of the CP8-CRM 97 conjugate in the above-mentioned murine bacteremia model was evaluated after S. aureus PFESA0268 (type 8) attack I49159.doc -84- 201110977 toxic (Figure 5 ) showed a significant reduction in bacteremia (p = 〇 0308). For the study, serotype 8 capsular polysaccharide-CRM97 was formulated with 丨〇〇叩aip〇4 by subcutaneous injection at weeks 0, 2, and 4. 6 to 8 weeks old Swiss Webster mice with physiological saline (Swiss Webstei&gt; actively immunized with ^) (η-3 0) and challenged with S. aureus PFESA026 8 (type 8) by intravenous route at week 6. The pre-challenge experimental line was in mice. The age reached after the second vaccination was carried out in order to optimize the dose of the challenge strain. The survival study was statistically evaluated by Kaplan-Meier analysis. Example 7. Immunization with native and chemically modified serotype 8 capsular polysaccharide conjugates The modulating activity of the serum of the mouse was determined by using the PFESA0005 strain, and the serotype 8 capsular polysaccharide high titer mouse serum (n=5) was compared from the immunization study to compare the opsonic activity β 〇pA results (Table 10). The conjugate produced by the combination of the native ash-clear type 8 capsular polysaccharide produces a modulating antibody in mice. It is worth noting that the de_〇Aci clear type 8 capsular polysaccharide conjugate is immunogenic in mice. However, the antibodies produced in this assay were not conditioned. The OPA titer was reported as the reciprocal of the dilution at the 40% kill rate observed. 149159.doc -85· 201110977 Table 10: Primary serotype 8 pods Membrane polysaccharide-immunized mouse serum compared to de-O-Ac Serotype 8 capsular polysaccharide mice immunized serum -CRM conditioning activity

De-O-Ac serotype 8 capsular polysaccharide-CRM serotype 8 capsular polysaccharide-CRM Week 0 serum OP titer Week 8 serum OP titer Week 0 serum OP titer Week 8 serum OP titer &lt ;50 &lt;50 50 150 &lt;50 &lt;50 &lt;50 1350 &lt;50 &lt;50 &lt;50 450 &lt;50 &lt;50 &lt;50 1350 &lt;50 &lt;50 &lt;50 4050 Example 8: The serotype 8 conjugate antiserum can be inhibited by the addition of native serotype 8 capsular polysaccharide to kill the S. aureus strain in order to confirm the specificity of killing, in native serotype 8 capsular polysaccharide or unrelated pneumococcal polysaccharide ( The opsonophagocytic assay was performed in the presence of Pn 14 poly), essentially as described above. The results (Table 11) show that the presence of native serotype 8 capsular polysaccharide in the reaction mixture inhibits opsonophagocytic killing of S. aureus PFESA0286 (type 8). These results confirm that the opsonophagocytic killing of immune sera is mediated by capsular specific antibodies. Table 11: Adding serotype 8 serotonin polysaccharide can inhibit the modulating of immune sera against S. aureus phagocytic killing monkey serum sample OPA titer WkO &lt;50 Wk8 4050 02D133 WkO + 20 pg CP8 poly &lt;50 Wk8 + 20 μβ CP8 poly &lt;50 WkO + 20 pg Pn 14 poly &lt;50 Wk8 + 20 pg Pnl4 poly 4050 149159.doc -86- 201110977

WkO &lt;50

Wk8 4050 A4N122 WkO + 20 μδ CP8 poly &lt;50

Wk8 + 20 pg CP8 poly &lt;50

WkO + 20 pg Pnl4 poly &lt;50 __Wk8 + 20 pg Pnl4 poly__1350 Summary All binding chemistry resulted in a serotype 8 capsular polysaccharide covalently linked to the carrier protein CRM197. There was no significant difference in the free sugar, serotype 8 capsular polysaccharide: protein ratio and the yield of the conjugate produced by such methods. Example 9: Preparation of S. aureus serotype 5 capsular polysaccharide In this example, the production of S. aureus serotype 5 capsular polysaccharides of various molecular size ranges is described. The structure of the S. aureus serotype 5 capsular polysaccharide repeating unit is shown in Fig. 6. The methods described herein are effective to produce serotype 5 capsular polysaccharides having molecular weights ranging from about 20 kDa to 800 kDa. High molecular weight serotype 5 capsules with molecular weights ranging from 50 kDa to 800 kD a can be isolated and purified by appropriate selection of conditions. For use in immunogenic compositions, serotype 5 capsular polysaccharides having molecular weights ranging from 70 kDa to 300 kDa, 70 kDa to 150 kDa, and many other desirable ranges can be isolated and purified. Based on the growth characteristics and the amount of capsule produced, strain PFESA0266 was selected to produce a serotype 5 film polysaccharide. In order to produce a serotype 5 capsular polysaccharide, the strain PFESA0266 was grown in a complex medium consisting mainly of a carbon source (lactose or sucrose), hydrolyzed soy flour as a nitrogen source and a trace metal. The strain is grown in a bioreactor for 2 to 5 days. The fermentation of the PFESA0266 strain was carried out as detailed above. At the time of collection, the OD6QG of the 149159.doc -87- 201110977 nutrient was 7.38. The culture was autoclaved for 1 hour, and after cooling, the culture was treated as described above to separate cells from the supernatant material. Approximately 1 L of each was filtered and the supernatant and cells were concentrated. Samples were removed to test the amount of staphylococcal enterotoxin B (SEB) in the culture prior to autoclaving. The concentration of SEB in the mash was 15-20 ng/ml in the presence of 0.05% polysorbate 80. Previous experiments showed that the hot-pressed culture for 1 hour reduced the SEB content to less than 0.1 ng/ml, which is below the detection limit of the TECRA kit. The diafiltered ethanol-separated polysaccharide was loaded onto a q-Sepharose Aec column&apos; and was eluted with a linear gradient of NaCl as described above. The fractions were analyzed by 〇- 醯 醯 analysis and two-way immunodiffusion test (testing for the presence of serotype 5 polysaccharide) and phosphate analysis (analysis of the presence of teichoic acid). The presence of the serotype 5 polysaccharide was detected in the fractions 60 to 105 (Figs. 7 to 7B). In order to reduce the contamination of the teichoic acid, the fractions 60 to 85 were pooled and any residual teichoic acid was oxidized with sodium metaperiodate to remove it by diafiltration against distilled water. The serotype 5 membranes used to prepare the conjugates were purified by two different methods that depend on high temperature and low pH to affect the release of the membrane from the cells and reduce the molecular weight of the polysaccharide. The molecular weight obtained depends on the time, temperature and positive value of the hydrolysis step. The serotype 5 smiley membrane was characterized using the technique described in Table 1 above. Pure polyenzymes with low levels of protein, nucleic acid, peptidoglycan and teichoic acid contaminants can be obtained by the method described below. In the first method, after the capsular polysaccharide is released from the cell leg and the molecular weight is lowered by 149l59.doc * 88 - 201110977, the preparation is treated with an enzyme mixture (such as ribonuclease, deoxyribonuclease, lysozyme and protease) to digest Impurities. After incubation, residual impurities were precipitated by the addition of ethanol (final concentration of about 25%). After removing residual ethanol, the capsular polysaccharide-containing solution was loaded on an anion exchange column (Q_Sepharose) and dissolved with a linear salt gradient. The fractions containing the capsular polysaccharide were pooled and treated with sodium metaperiodate. This treatment resulted in oxidative hydrolysis of residual teichoic acid contaminants, but did not affect serotype 5 capsular polysaccharides. The reaction was quenched by the addition of ethylene glycol. The material was concentrated and dialyzed against distilled water (dH2) to remove any residual reagents and by-products. The first method is used to produce capsular polysaccharide without the use of enzymes to digest various cell-derived impurities. In this method, after the capsular polysaccharide is released from the cells and the amount of the knives is lowered, the hydrolyzed fermentation broth is clarified by microfiltration, followed by ultrafiltration and diafiltration. The solution was treated with activated carbon to remove impurities. After carbon treatment, the material was treated with sodium metaperiodate to oxidize the residual teichoic acid, followed by quenching with propylene glycol. The material was concentrated and dialyzed against distilled water to remove any residual reagents and by-products. Pure capsular polysaccharides having low levels of protein, nucleic acid and phosphobiochemical contaminants can be obtained by using any of the methods to produce a preparation. The method can be used to produce a polysaccharide having a particular desired high molecular weight range by controlling the hydrolysis conditions. Examples of capsular polysaccharides obtainable by the methods described herein are shown in Table 12 below. The purified serotype 5 capsular polysaccharides of the batches are of high purity, as indicated by the no-wall acid (TA), peptidoglycan and low residual protein. See Tables 12 and 13. The molecular weight ranged from 132.7 kDa to 800 kDa, and the purification was _ highly Ο-acetylated (in the range of 90% to 100%) and passed through 1〇〇% N_ 醯 U9159.doc • 89 - 201110977. The purified yield of serotype 5 capsular polysaccharide was 39% to 63%, and the purified serotype 5 polysaccharide had molecular sizes ranging from 35 kDa to 65 kDa (see Table 12). The degree of contamination of the phosphonic acid (TA) is acceptable, and the residual protein and nucleic acid content are also within acceptable limits. The NMR spectrum of the serotype 5 polysaccharide was consistent with those reported in the literature. Table 12: Serotype 5 capsular polysaccharide characterization sample CP5 yield MW protein nucleic acid 〇-acetyl group mg % (kDa) % (w / w) (R / mol) (260 nm scan) % (w / w) NMR (%) 1 101 39 47 0 0.5 94 2 91 48 65 1.2 2.5 96 3 578 63 35 2.5 0.7 75 Table 13: Characterization of other serotypes 5 capsular polysaccharides. O-Ethyl N-acetonitrile sample MW (kDa) CP5 (mg/ml) NMR (%) NMR NMR (°/〇) 1 800.1 3.164 100 by 100 2 132.7 1.172 90 by 100 3 335.4 0.975 90 100 4 366.8 0.865 90 By ND ND = no molecular weight selection of capsular polysaccharides was selected: kinetic analysis showed that the methods described herein produced capsular polysaccharides with a broad molecular weight range. First, a larger polysaccharide is produced from the bacterial cells, followed by selection of the desired molecular weight range, followed by purification by controlling the pH of the heating and hydrolysis steps and heating conditions. The heat treatment of the Staphylococcus aureus fermentation broth is a processing step between the fermentation and the capsule 149I59.doc -90- 201110977 polysaccharide recovery. The culture medium is treated for a specific period of time. Kill cells, inactivate intestinal toxins, and reduce to the desired size. The reduction in molecular weight is the slowest among these targets. It is inevitable to achieve other goals. This treatment step uses heat to adjust the pH to a low pH value. The goal of heat treatment is to release the cell-bound polysaccharide and to treat the molecular weight in the process required for this step. Therefore, the heat treatment is determined during the treatment time considered. Select the pH value and pulse condition of various molecular weight ranges of the capsule to make the 5 Lf music. The sterilized fermentation broth is transferred to the broth by means of a screw spring. Using the search speed of about 鸠犷 (4), adjust the culture solution ρ· with han sulfuric acid. Next, the culture solution was allowed to warm to a value of X疋. Once the temperature reaches the set point, the heat treatment timer begins. When the desired treatment time is reached, the solution 4 is cooled to room temperature. Samples of treatment orders were obtained and the polysaccharide concentration and molecular weight were determined by HPLC and SEC-MALLS systems, respectively. Molecular weight (MW) data were used for kinetic analysis. The MW distribution in the assay is measured with time 璧 8A as shown in Figures PH 3.5, 4.0 and 5.0. The kinetic analysis of the weak hydrazine hydrolysis of the polysaccharide was carried out using purified serotype 8 capsular polysaccharide obtained from the treatment. The purified polysaccharide solution was adjusted to the desired pH with sulfuric acid for experimental use. Transfer about 5 solutions to each of the 15 red centrifuges. Place the centrifuge tube in an oil bath equipped with a precision temperature control system. Remove the centrifuge tube every predetermined time and quench it in an ice bucket. At the end of the experiment An aliquot of i M Tris buffer 7.5) was added to the sample to adjust the pH back to about 7. Samples were analyzed by the SEC_MALLS system. Na data is used for kinetic analysis. The effect of temperature over time on the MW distribution of 149159.doc •91 · 201110977 was measured at pH 4 5 . See Figure 8B. Results As shown in Figure 8A, a lower pH value is more effective in reducing the molecular weight of the polysaccharide. In this example, a molecular weight range of from about 300 kDa to about 600 kDa can be produced using pH 5, 95 ° C for 15 minutes to 120 minutes. See Figure 8A. Similarly, the pH range of 200 kDa to 400 kDa can be produced by selecting pH 4·5, 95 ° C for 15 minutes to 120 minutes. In addition, the selection of pH 4.0, 95 ° C, for 15 minutes to 120 minutes can produce a molecular weight range of polysaccharides from 120 kDa to 300 kDa. As shown in Fig. 8B, the higher the temperature, the faster the hydrolysis rate and the wider the molecular weight range of the polysaccharide produced. Expressed in another way, a lower temperature of 55 is used at the same pH. 〇 (relative to 95. 〇 produces a narrower molecular weight range. In addition, Figure 4 shows the correlation between the molecular weight of the purified serotype 5 capsular polysaccharide and the weak acid (pH 4.5, 95 ° C) hydrolysis treatment time. The final product of the recovery treatment detailed above. As shown in Fig. 4, prolonging the heat treatment of S. aureus PFESA0266 strain at pH 4·5 can produce a serotype 8 seromembrane multi-enzyme with a molecular weight of less than * The heat treatment time of yang 4 5 can produce more than the molecular weight of the Qiqing type 5 laughing film. The length of the heat treatment is PH 4,5, and the molecular size of the serotype 5 capsule is about 90 kDa. Up to about 22 〇 kDa. As shown in Figure 4, the correlation between the heat treatment time at low pH and the molecular size of the purified serotype 5 inch membrane (4) can be used to evaluate the production of purified polysaccharides with a molecular weight range. The required treatment time. 149159.doc •92· 201110977 As shown above, serotype 5 capsules can be produced, released and purified from 2〇kDa to over 500 kDa. The method can be used to produce Specific height A capsular polysaccharide having a molecular weight range, such as shown in Table 14. A polysaccharide having a relatively narrow molecular weight range (wherein peak molecule I is in the range of 63 kDa to 142 kDa) represents a process obtainable by the method described herein. a fully characterized molecular weight range. Polysaccharides with a particularly advantageous high molecular weight range (in the range of 70 kDa to 300 kDa or 7〇kDa to 15〇kDa) are suitable for immunization by binding capsular polysaccharides to carrier molecules or proteins. The original composition. The conditions for producing CP5 capsular polysaccharide having a molecular weight ranging from about 1 〇〇 to 140 kDa are as follows: 9 yc, pH 4 5, lasting 5 minutes. However, the difference between ρ η value, temperature and time Also: a CP5 molecule having a molecular weight ranging from about 100 kDa to 140 kDa. Table Η: Producing a serotype with a specific high molecular weight range. capsular polysaccharide serotype 5 capsular polysaccharide MW (kDa)

3 5 7 9 10

142 108 142 108 ND ND 63 72 74 63 ND 149159.doc -93· 201110977 Example 10: Serotype 5 capsular polysaccharide binding to crm197 This example describes the production of S. aureus serotype 5 capsular polysaccharide _ crm197 conjugate Methods and characterization analysis. Different binding chemistry techniques have been developed for binding S. aureus serotype 5 capsular polysaccharide to this carrier protein. For example, the use of PDPH (3_(2-pyridyldithio)-propionamidine) binding can produce a covalent thioether bond between the CP and the carrier protein; whereas CDT (1,1-carbonyl-di-) is used. 1,2,4-triazole) binding produces a single carbon or zero carbon linker between the capsular polysaccharide and the carrier protein.

Binding of cytosolic polysaccharides to CRMi97 by PDPH binding chemistry is a multi-step process involving activating polysaccharides, removing thiol protecting groups, purifying activated polysaccharide intermediates, and activating and purifying CRMm proteins. 'And the activating components are combined, followed by purification. The introduction of a thiol group-containing linker into the polysaccharide and introduction of the haloacetic acid group into the CRM!9? protein carrier allows the golden yellow grape spheroid serotype 5 laughing membrane to be ligated to the protein carrier via a thiol bond. The bromoethenyl group is introduced into the CRM]97 protein by a reaction of an amine group with N-hydroxybutylimine of bromoacetate. In order to produce a thiolated polysaccharide, the carbodiimide-activated formate group of N-ethyl decylamino mannitol in the polysaccharide is coupled to a thiol-reactive doped bifunctional linker 3-(2) - Pyridyl disulfide) - a sulfhydryl group of propyl hydrazine (PDPH). The thiol of the pj)PH thiolated polysaccharide purified by DTT and produced by TT reduction on the Sep had ex G25 column is reacted with the bromoethane group of the activated protein to thereby replace the polysaccharide with the carrier by bromine A covalent thioether bond is formed between the proteins. The unreacted bromoacetyl group is "capped" with cysteamine hydrochloride (2-aminoethylthiol hydrochloride). The reaction mixture was then concentrated and the dialysis transition. Residual unknot 149159.doc -94- 201110977 The bromoethane group was capped with cysteamine hydrochloride to ensure that no reactive bromoethylidene group was present after binding. Thus, after bromine displacement, a covalent bond is formed between the thiol end of the cysteamine and the acetamidine group on the amino acid residue. 1. Sulphurization of S. aureus serotype 5 capsular poly Sf with PDPH: The polysaccharide was first activated by thiolation of PDPH. Mix the polysaccharide with freshly prepared PDPH stock solution (250 mg/mL in DMSO), EDAC stock solution (90 mg/mL in distilled water) and MES buffer stock solution (0.5 Μ, pH 4.8 5) The final solution contained 0.1 M MES, and 2 mg/mL and 4 mg/mL capsular polysaccharide, while maintaining a 1:5:3 polysaccharide:PDPH:EDAC weight ratio for serotype 8 capsules. Incubation was carried out for 1 hour at room temperature, followed by dialysis against 4 volumes of distilled water 4 times at 4 ° C to 8 ° C using a 3500 MWCO dialysis apparatus to remove unreacted PDPH. The PDPH-linked polysaccharide was produced using a 0.2 M DTT solution and incubated for 3 hours at room temperature or overnight at 4 °C to 8 °C. Sephadex G25 resin and distilled water were used as the mobile phase, and excess DTT and reaction by-products were separated by self-activated sugar by SEC. The thiol group of the dissolved fraction was analyzed by DTDP analysis, and the thiol-positive dissolved fraction dissolved in the vicinity of the void volume of the column was collected. The dissolution fraction was analyzed by PAHBAH and 0-acetamidine analysis to determine the degree of activation, and the degree of activation expressed as the molar percentage of the thiol group-containing repeating unit (molol concentration of thiol / molar concentration of repeating unit) . The activated polysaccharide was lyophilized and stored at -25 ° C until binding was required. The results of thiolation of the serotype 8 polysaccharide by PDPH are shown in Table 15. The serotype 5 polysaccharide has an activation degree in the range of 11% to 19%, corresponding to about 1 linking molecule per 10 capsular polysaccharide repeating units to every 5 repeating units. 149159.doc -95- 201110977 1 connection molecule. Table 15: Activation of serotype 5 capsular polysaccharide by PDPH-reproducibility study serotype 5 polysaccharide-PDPH activation degree (% Msh/Mru) scale rag yield mg (%, w/w) 1 11 23 19.6 (85 2 13 30 28 (93) 3 19 30 23(77) 4 15 32 29 (90) 2. Carrier protein activation: The carrier protein is activated independently by bromination. CRM197 was diluted to 5 mg/mL with 10 mM phosphate buffered 0.9% NaCl (pH 7) (PBS), followed by pH 7.0 with 0.1 Μ NaHC03. iV-hydroxybutylimine (BAANS) bromoacetate was added at a CRM 丨 97:BAANS ratio of 1:0.25 (w:w) using a BAANS stock solution of 20 mg/mL DMSO. The reaction mixture was incubated at 4 ° C to 8 ° C for 1 hour, followed by purification on Sephadex G-25 using SEC. The purified activated CRM197 was analyzed by Lori analysis to determine the protein concentration, followed by dilution to 5 mg/mL with PBS. Sucrose was added as a cryoprotectant up to 5 ° / 〇 wt / vol, and cold; East activated protein and stored at -25 ° C until binding is required. The bromoacetylation of the aminic acid residue of CRM197 is extremely coherent, thereby activating 19 to 25 of the 39 lysines available for use (see Table 16). This reaction produces activated proteins in high yield. 149159.doc -96- 201110977 Table 16: Yield and extent of CRMm bromoacetamidine Deactivated lysine (n=) scale yield---------- (%w/w) 1 24 23 85 2 20 38 87 3 19 35 77 4 22 35 94 5 23 35 87 6 25 48 1〇4 3. Coupling reaction: : Activated capsule multi-riding and --- activation of the carrier 'even if both Compounded in the binding reaction. The lyophilized and thiolated polysaccharide was dissolved in (M6 oxime acid salt (pH 8.95), mixed with the solution of sulphuric acid CRMw and distilled water, so that the final solution contained 〇1 river borate, 1:1 weight ratio CRMm: Polysaccharide and 2 mg/mL serotype 5 capsular polysaccharide. Incubate for 16 hours to '24 hours at room temperature. II by using 135 mg/mL cysteamine dissolved in M-Putolate (pH 8.95) In a stock solution, cysteamine hydrochloride is added at a CRMm··cysteamine ratio of 1:2 (wt/wt) to cap the unreacted bromoethylidene on the protein and incubate at room temperature for hours. Purification of capsular polysaccharide _CRM|97 conjugate (conjugate) by 5 diafiltration with respect to 〇9% Naci using a 100K polyethersulfone ultrafilter. Reproduction of serotype 5 capsular polysaccharide sulphur by PDPH The results of alcoholization studies show that the degree of activation is in the range of 11% to 19%, corresponding to every 1 (H@cp repeat unit is linked to about 1 linker molecule to every 5 repeat units connected to a linker molecule 0 by CDT binding chemistry The method provides a serotype 5 capsular polysaccharide and CRM... in combination with CDT to provide a step-by-step method, which is mostly in the anhydrous environment _s(7) 149159.doc •97· 2011 10977 is activated to form a triazolyl decanoate moiety having a usable hydroxyl group and a thiol group having a carboxylic acid. The sitting or thiol tris. The addition of a protein carrier (containing in DMSO) causes nucleophilic displacement of the amine acid. Triazole and the formation of amino phthalate linkages (for reactive hydroxyl groups) and guanamine linkages (for active carboxylic acids). The reaction solution is diluted 1 〇 in aqueous solution to prepare for tangential flow filtration purification. The chemical method produces a serotype 5 capsular polysaccharide covalently linked to a carrier protein, according to the following instructions: the presence of sugars and proteins in the fractions of size exclusion chromatography and the blocking with glycolaldehyde or cysteamine hydrochloride Amino acid analysis of salt-terminated conjugates. Preparation of serotype 5 capsular polysaccharides Multi-batch conjugates with molecular size ranging from 20 kDa to 40 kDa (prepared by PDPH and CDT chemistry) are summarized below In Table 17. The ratio of free capsular polysaccharide, polysaccharide·protein ratio and yield of the combination produced by the combination of chemical methods is not significantly different. The antigenicity of the combined serotype 5 capsular polysaccharide is not due to binding. change As shown by the consensus between the conjugate and the native polysaccharide. Table 17: Characterization of serotype 5 capsular polysaccharide-crm197 protein prepared by two binding chemistry methods. Yield yield chemical method (%) ( %) CDT 19-27 35 Output ratio 0.5-0.8 Molecular size (MW or ^ Free egg modified Kd (% &lt; 0.3), more m } White matter (%) Amino acid sugar / protein)) 10-40 &lt;;1 18-22 38/61 to 76/74 PDPH 26-52 40-99 0.4-1.0 23-50 ND ND 7.5&gt;&lt;105 to 2.3&gt;&lt;106 ND = not detected as shown above The methods described herein can be used to produce capsular polysaccharides having a particular desired high molecular weight range. A conjugate was prepared from a serotype 5 capsular polysaccharide having a pre-selected high molecular weight formula 149159.doc 98- 201110977 which can be filtered and purified for use in an immunogenic composition. Table 1 summarizes the analysis of serotype 5 capsular polysaccharide conjugates wherein the serotype 5 capsular polysaccharide has a molecular weight in the range of from about 92 kDa to about 119 kDa and is activated with triazole (CDT). The molecular weight of the resulting conjugate is in the range of 1 533 kDa to 2656 kDa. The number of bound lysines in each CRM197 ranged from a high value of 22 to a low value of 15. The free capsular polysaccharide ranges from a high value of 18% to a low value of 11%. Table 18: Conjugate polysaccharide MW of serotype 5 capsular polysaccharide with pre-selected molecular weight range, by SEC-modified MW (kDa) yield (%) free sugar (%) MALLS (kDa) Amino acid 1 121 63 11 2130 19 2 92 72 16 1533 22 3 119 74 14 2656 15 4 115 63 18 1911 15 Both binding chemistries produce a serotype 5 capsular polysaccharide covalently linked to a carrier protein. There was no significant difference in the ratio of capsular polysaccharide to serotype 5 capsular polysaccharide: protein ratio and yield between the two methods. Example 11 Complicated Method Compared to the One-Step CDT Method As described above, the method for producing the immunogenic conjugate of the present invention comprises covalently binding a capsular polysaccharide to a carrier protein using a binding chemistry method, including 0 Agaridine (1,1-carbonyl-di-1,2,4-triazole) or? 〇? Other 3-(2-pyridyldithio)-propanoid). Using CDT to produce a single carbon or zero carbon linker between the capsular polysaccharide and the carrier egg 149159.doc -99· 201110977 white matter, and using PDPH to produce a covalent sulfur-containing five between the membrane polysaccharide and the carrier egg self-mass Carbon linker. The PDPH-based approach is a multi-step process involving activation of the brew, removal of the polythiol protecting group, purification of the activated multi-compartment, activation and purification of the protein carrier&apos; and binding of the activating component followed by purification. Here, &gt; gold color grape ball gjk clear type 5 capsule multi-powder and η and carbodiimide are reacted in an organic solvent such as DMS0 to produce a pDpH-linked multi-enzyme. The PDPH-linked polysaccharide reacts with a reducing agent to produce an activated polysaccharide, which is then purified. The carrier protein is reacted with desert acetic acid in an organic solvent to produce an activated carrier protein, which is then purified. The purified activated serotype 5 polysaccharide is then reacted with the purified activated carrier protein to produce a serotype 5 polysaccharide: carrier protein conjugate. In contrast, the CDT-based method is a one-step method in which a capsular polysaccharide is activated in an anhydrous environment (also mDMS0) to form a triazolamide moiety having a hydroxyl group available and a mercapto imidazole having a carboxylic acid or Mercapto triazole moiety. Addition of a protein carrier (containing MDMS〇f) causes nucleophilic displacement of the imidazole or triazole from the amine acid and formation of a urethane bond (for the reactive hydroxyl group) and a guanamine bond (for the active carboxylic acid)' to allow for the combination of "one step The form is carried out. Therefore, two methods based on CDT have been developed: a more complicated method and a simpler one-step method. In a more complex method, S. aureus serotype 5 capsular polysaccharide is combined with imidazole or triazole' followed by reaction with CDT in an organic solvent such as DMSO and about 0.2% w/v water to produce activated serotype 5 Polysaccharide. Purification of the activated serotype 5 polysaccharide&apos; is then reacted with the carrier protein in an organic solvent to produce a serotype 5 polysaccharide: carrier protein conjugate. One 149159.doc •100- 201110977 The footwork is similar to the complex method except that the activated serotype 5 polysaccharide is not purified prior to reaction with the carrier protein. CDT Complex Method Activated serotype 5 capsular polysaccharide: Serotype 5 capsular polysaccharide was mixed with 10 gram of triazole/gram serotype 5 capsular polysaccharide and lyophilized. The resulting cake was dissolved in DMSO at 2.0 mg serotype 5 capsular polysaccharide per ml. The water content was measured and adjusted to 0.2%. A freshly prepared CDT stock solution (100 mg/mL in DMSO) was added until the amount of CDT was 20 times the molar excess of CP5. Alternatively, the amount of CDT added can be adjusted to achieve a higher or lower degree of activation. Hold at 23 ° C for 30 minutes. Purification of activated serotype 5 capsular polysaccharide: A solution of activated serotype 5 capsular polysaccharide (ACP5) was poured into 25 volumes of water to remove excess CDT. It was concentrated to about its original volume on a 10 kDa PES film at about 1 mg/cm2 and diafiltered against at least 10 volumes of water. This step is completed in less than 4 hours. The diafiltered material was mixed with 10 g of triazole/g raw serotype 5 polysaccharide and lyophilized. Preparation of lyophilized CRM: CRM was dialyzed against a 10 kDa PES membrane in a constant volume relative to at least 10 volumes of 0.4% NaCl/5% sucrose. The protein concentration was measured, and sufficient diafiltration buffer was added to bring the protein concentration to 5.0 g/L, thereby obtaining a NaCl/CRM weight ratio = 0.8. Freeze dry CRM. Binding: Activated, diafiltered serotype 5 capsular polysaccharide was dissolved in DMSO at 1 mg/mL. Add 100 mM borate solution until 2% v/v is reached. The CRM was resuspended at 2 mg/mL and, when completely dissolved, was combined with the ACP5 solution 149159.doc -101 - 201110977. Allow to react at 4 ° C for 20 hours. The conjugate reaction solution was poured into 24 volumes of 5 mM borate (pH 9.0) and stirred at room temperature for 1 hour. It was then adjusted to pH 7.5 with hydrazine, 5 Μ phosphate buffer (pH 6.5). It was filtered through a 5 micron filter and concentrated to the original volume on a 3 〇〇 kDa PES film at a load of about 1 mg/cm 2 and diafiltered against at least 10 volumes of water. The resulting concentrate was filtered through a 〇 22 μm filter and stored at 2 ° C to 8 ° C. CDT-step CRM 9 matrix exchange: diafiltration of CRMi97 to exchange about 1 mM phosphate / 80 mM NaCL / 15% sucrose whole matrix (pH 7) into 5 mM imidazole / 0.72% NaCl / 15 mM xin --β-D-glucoside glucoside (pH 7). Exchange can remove phosphate and sucrose that are detrimental to binding and will limit the amount of sodium chloride delivered to the bond. The octyl_p_D_glucopyranoside is added to prevent the formation of particles after sterile filtration. Exchange of (^]^197 matrix by tangential flow, relative to 5 mM saliva/0.72% NaCl/15 mM octyl-β-D-glucopyranoside (pH 7) (via 10 dialysis Filtration volume multiple 'use 10K MWCO PES film, retention concentration is about 4 mg / mL,) typical film limit is 2 g / ft 2, and the final target crm197 concentration in the matrix is 6 mg / mL. CRMi97 at 2 ° C to Storage at 8 ° C. Activation / binding: S. aureus serotype 5 capsular polysaccharide activation / binding method consists of the following steps: 1) compounding the polysaccharide; 2) shell freezing and lyophilization CRM] 97 and compounding Polysaccharide; 3) Dissolve lyophilized polysaccharide and; 4) Activate polysaccharide; 5) Combine activated polysaccharide with CRMm; and 6) Purify knot J49159.doc -102- 201110977 Compound (dilution, diafiltration, sterile filtration) ). The polysaccharide is combined with 10 grams of 1,2,4-triazole excipient per gram of polysaccharide. The excipients are added to the polysaccharide in powder form and a solution is obtained after mixing for less than 15 minutes at ambient temperature. The combined polysaccharide and CRM197 were shell-frozen using a -75 °C ethanol bath, respectively. The volume per 1 L of bottle is approximately 500 mL. To dissolve the polysaccharide, DMSO is added to individual freeze-dried vials of the polysaccharide to obtain a suspension which is then transferred to an activation/binding reaction vessel for heating. DMSO was added until a concentration of 2 g/L was obtained. A clear solution was obtained after 5 to 10 minutes of mixing. To dissolve CRM197, DMSO was added to individual lyophilized vials containing CRM197 to obtain a suspension, which was then transferred to a second vessel for mixing. DMSO was added until a concentration of 2 g/L was obtained. A clear solution is usually obtained in less than 15 minutes. A card-fee analysis was performed on the polysaccharide/DMSO solution to determine the moisture content. CDT was prepared as a 100 mg/mL solution in DMSO and added in a 5 molar excess to the type 5 polysaccharide (20 molar equivalent CDT was used in the complex method and 5 mole equivalent CDT: CP5 in one step). The CDT solution was continuously added over 5 minutes at 23 ° C + 2 ° C with mixing. The reaction was carried out at 23 ° C ± 2 ° C for a minimum of 30 minutes. The reactants were sampled to determine the degree of activation (UV 220/205 nm), followed by the addition of 100 mM sodium borate (pH 9) to obtain 1.5 °/. Aqueous solution. Next, the reaction solution was stirred at 23 ° C + 2 ° C for a minimum of 30 minutes. In order to bind the activated polysaccharide to CRM197, DMSO was added to obtain a reaction concentration of 0.55 mg/mL. CRM197 dissolved in DMSO is then added to the activated polysaccharide solution under mixing 149159.doc • 103-201110977. Stir the reaction for at least 16 hours under phantom ^^^. The reaction solution was diluted 1 〇 with 5 mM sodium tetraborate (ρΗ 86) to obtain a final diluted solution (pH (4). 2). The solution was stirred at 23 t ± 2 t for a minimum of 4 hours. The diluted solution was passed through a 5 μm filter and concentrated to 2 g/Li target retention/length. Tangential flow filtration was carried out using 5 mM succinate (ppj 7), using a diafiltration filter volume of 20 diafiltration filters, using a 300K regenerated cellulose film. The typical diaphragm is limited to 1 g/ft2. The purified conjugate was passed through a 〇 22 μm filter ' and at 2. (: Store at 8 ° C. Example 12: Combining serotype 5 capsular polysaccharides using one-step and complex binding methods This example shows that capsular polysaccharides with pre-selected molecular weight ranges can be used in one-step or complex methods. Combining. Firstly, the bacterial cells produce larger vinegar' and the molecular weight range can be controlled by the pH of the hydrolysis process of Example 9 and heating. In this example, the molecular weight of the selected gold capsular polysaccharide is about 90 kDa. Up to 8 batches in the range of about 140 kDa, and combined using triazole (CDT) activation in the one-step or complex method described above. See Table 19. The molecular weight of the resulting conjugate is in the range of 1125 kDa to 2656 kDa. The number of bound lysines in CRM ranges from a high value of 22 to a low value of 15. Free sugars range from a high value of 23% to a low value of 11%. 149159.doc -104- 201110977 Table 19: Use 90 kDa to 140 kDa Method for the preparation of capsular polysaccharides by serotype 5 capsules conjugate method. Polysaccharide MW (kDa) Sugar yield (%) Free sugar (%) MW, one step by SEC-MALLS (kDa) lysine 1 142 93 11 1932 19 2 108 93 14 1117 20 3 142 85 17 1609 15 4 108 86 2 3 1125 15 Complex method 1 121 63 11 2130 19 2 92 72 16 1533 22 3 119 74 14 2656 15 4 115 63 18 1911 15 Example 13: Serotype 5 capsular polysaccharide conjugates continue to show protection in the murine pyelonephritis model The role of the serotype 5 capsular polysaccharide conjugate in protecting the mouse in the pyelonephritis model was evaluated. The count of bacteria in the blood of mice infected with S. aureus was significantly lower than that of the control group immunized with PBS. All six individual studies showed a significant reduction in cfu/ml in the kidneys of immunized animals (Figure 9). When these studies were pooled for meta-analysis, the overall significance of the studies was generally increased. To less than 0.0001. The data show that renal colony formation continues to decrease after active inoculation with the capsular polysaccharide conjugate. Example 14: Serotype 5 capsular polysaccharide conjugate prepared by different binding chemistry methods protects mice from experimental infection Active immunization studies were performed in a murine pyelonephritis model using a serotype 5 capsular polysaccharide conjugate prepared by PDPH or CDT chemistry. The capsule was 149159.doc -105- 201110977 The method of binding to CRM197 is described above. The results show that both conjugates reduce colony formation in mice compared to sham-immunized animals (Table 20). Table 20: Effect of PDPH versus CDT binding on prevention of S. aureus challenge in a pyelonephritis model. Study number antigen strain/dose logCFU/kidney significance study 1 Physiological saline + Α1Ρ〇4 PFESA0266 5.53 ± 1.90 — 1 meg CP5-CRM (PDPH) +AIPO4 2x108 3.01 ± 1.83 p&lt; 0.001 Study 2 1 meg CP5-CRM ( CDT) +AIPO4 Physiological saline + aipo4 PFESA0266s 1.67±0_23 6.17 ±1.76 p&lt; 0.0001 1 meg CP5-CRM (PDPH) +AIPO4 2.7x10s 3.06 ± 1.69 p &lt; 0.0001 1 meg CP5-CRM (CDT) +AIPO4 1.87 ± 0.69 p &lt; 0.0001 Example 15: Active immunization with serotype 5 capsular polysaccharide conjugates Rats were protected in a rat endocarditis model with the CP5-CRM197 PDPH conjugate for 4 studies. After challenge with S. aureus PFESA0266, in two-thirds of the experiments, the serotype 5 capsular polysaccharide conjugate significantly reduced the recovery CFU in the heart and kidney (Table 21). In the third study, the geometric mean titer (GMT) of anti-CP5 titers was the lowest of the three experiments, but only slightly lower than previous experiments. 149159.doc •106- 201110977 Table 21: Serotype 5 capsular polysaccharide-CRM197 immunization reduced CFU in a rat endocarditis model.

Log restore CFU significant GMT immunogenic composition challenge strain / dose heart kidney kidney kidney CP titer 1 meg CP5-CRM PFESA0266 4.34 ± 1.78 3.92 ± 1.73 103,000 1 meg PP5-CRM 2.21xl08 efu 7_94 ± 0.78 6.77 ± 0_79 pO.OOl p&lt;0_05 1 meg CP5-CRM PFESA0266 4.43± 2.30 3.11±2.33 51,000 saline 6.5xl07 efu 5.63± 2.48 4.19±2.05 no 1 meg CP5-CRM PFESA0266 4.01± 2.49 3.90± 1.92 67,000 saline 4 〇χ108 efu 7.52± 1.38 6.52± 1.17 p&lt;0.0002 p&lt;0.0002 Example 16: HMW CP5 conjugate vaccine is more immunogenic in mice than LMW CP5 conjugate vaccine for murine pyelonephritis studies to assess different CP5 binding The immunogenicity and potency of the formulation. Two formulations were tested: the first formulation consisted of a combination of high molecular weight (HMW) CP5 (about 300 kD) and CRM197. The second formulation contains a combination of low molecular weight (LMW) CP5 (about 25 kD) and CRM]97. Three dose concentrations of the HMW vaccine, oj and 〇1 meg) were tested. The LMW vaccine was tested at 1 meg. Also included is a negative control group consisting of a polysaccharide conjugate vaccine. The vaccine is derived from a combination of S. pneumoniae and CRM] 97 (PP5). Polysaccharides were formulated with 22 meg Α1Ρ〇4 at weeks, 3 and 6 weeks, and challenged with S. aureus PFES A〇266 at week 8. The kidneys were collected 48 hours after challenge and the bacterial community was counted. Both vaccines were effective in generating an immune response&apos; and a decrease in CFU of S. aureus PFESA 266 was observed in the kidneys of mice at 1 pg HMW and LMW vaccine treatment groups. This reduction is dose dependent' as shown by the decrease in efficacy as the vaccine dose decreases (Figure 10). For the detection of HMW and LMW vaccine effects, the CFU readings are not sensitive enough. Therefore, the mouse serum was tested by opa. OPA titer was defined as the serum dilution required to kill 40% of the S. aureus strain PFESA0266 in the OPA assay. It can be seen that the OPA titer of the HMW vaccine is higher than that of the LMW formulation (Fig. 11). Example 17: A capsular polysaccharide conjugate comprising a high molecular weight polysaccharide exhibits greater immunogenicity than a conjugate comprising a low molecular weight polysaccharide. Non-human primate (NHP) was studied to assess immunity of different membrane conjugate formulations. Originality. Two formulations were tested at two different dose concentrations (2 ton and 2 〇 pg). The first formulation contained a combined high molecular weight (HMW) polysaccharide (about 130 kD) and CRMw. The second formulation contains a combination of low molecular weight (LMW) polysaccharides (about 25 kD) and CRM 丨97. Five primates in each group were vaccinated with either single dose of vaccine and immune titers were monitored before vaccination and two weeks after vaccination. The OPA titer is defined as the serum dilution required to kill 40% of the S. aureus strain PFESA〇266 in the Opa assay. Antibody titers were also monitored by ELISA. It can be seen that the activity of the HMW vaccine is stronger than that of the LMW formulation (Table 22), as evidenced by the antibody titer of the HMW vaccine as a multiple of the LMW vaccine. The 〇pA response rate of NHP receiving HMW vaccine was also higher (80% compared to 40%). Table 22. Immunogenicity of the H1VIW multi-audioconjugate vaccine was observed to be stronger than the lmw polysaccharide conjugate vaccine CP5-CRM197 dose (meg) / animal PD1* geometric mean OPA response rate (%) Shi HMW (125 kD) 20 32 ~ 8 (Γ 2 21 ~~80 LMW(25 kD) 20 3 &quot;~40 ' 2 8 40 I49159.doc -108- 201110977 * Increase the fold by comparing the CP5 ELISA titer and vaccination 2 weeks after inoculation The pre-existing titer ^^7^^ rate was calculated based on the increase in OPA titer produced by monkeys 2 weeks after inoculation of a single-dose vaccine. Each group contained $only rhesus macaques, and the vaccine was formulated with AlP〇4 (250 Micrograms/dose).___,. Example 18: Polysaccharide 〇· acetylation is important for inducing protective antibody responses against serotype 5 pod poly-conjugates in order to assess serotype 5 capsular polysaccharide 0-acetamidine Importance, remove the o-acetyl group (dOAc) of the native capsular polysaccharide and bind to CRM197 (dOAc-CRM197) using the PDPH binding chemistry discussed above. Parallel comparison of dOAcCP-CRM197 with CP5-CRM丨97. Efficacy in a murine pyelonephritis model. Immunization with a conjugate lacking 0-acetamido (dOAc CP5-CRM) failed to reduce kidney Recovering bacterial CFU. These data (Table 23) indicate that 〇_乙醯化 is important for inducing functional antibodies against CP5. Table 23: De-O-acetamidine gold-type 5 smiling membrane conjugates Immunization does not prevent mouse kidney colony formation. Numbered antigen strain/dose LogCFU/kidney significance study 1 1 meg PP5-CRM PFESA0266 3.89 ±2.24 11 _ _ 1 meg dOAc CP5-CRM 7χ108 4.20 Soil 1.75 1 meg CP5-CRM 1.75 ± 0.39 P value &lt;0.008 Study 2 Physiological saline PFESA0266 5.08 ± 1.96 1 meg dOAc CP5-CRM 2.4χ108 5.89 ± 1.29 1 meg CP5-CRM 2.93 ± 2.11 P value &lt; 0.02 Example 19: Using a single with known specificity Evaluation of the importance of 〇-acetamidine as a functional epitope of serotype 5 capsular polysaccharide by OPA for OAc + (CP5-7-l), OAc +/- (CP5-5-l) and OAc-(CP5-6-149J59.doc-109-201110977 1) Specific serotype 5 capsular polysaccharide monoclonal antibody against OP killing activity of type 5 strain PFESA0266 (Table 24). Monoclonal antibody against serotype 8 capsular polysaccharide (CP8-3-1 specific for CP8 OAc+) was used as a negative control. OAc-specific anti-CP5 mAb CP5-7-1 mediated S. aureus PFESA0266 Killed (Table 24). Furthermore, the monoclonal antibody CP5-5-1, which recognizes the epitopes shared by CP5 OAc+ and CP5 OAc, mediates the killing of the PFESA0266 strain. The monoclonal antibody specific for the epitope present on the serotype 5 OAc-capsular polysaccharide does not mediate the killing of the PFESA0266 strain. These results indicate that the 〇-acetyl thiol epitope on the serotype 5 capsular polysaccharide is necessary for inducing the functional activity of the serotype 5 specific antibody. The antibody needs to be functional, as measured by killing the cells in an animal efficacy model demonstrating that the antibody kills the bacteria or in the analysis of the killing cell killing. The functional killing effect may not use the analytical evidence of monitoring antibody production alone, and such an analysis does not indicate the importance of Ο-acetamidine in efficacy. Table 24: Monoclonal antibodies specific for Ο-acetylated (+) serotype 5 capsular polysaccharide and Ο-acetylated and De-O-acetylated (+/·) serotype 5 capsular polysaccharide Conditioning with S. aureus PFESA0266 (type 5) CP5-7-l (0-Ac +) (meg) 20 10 5 2.5 CP8-3-l (-control) (meg) 20 10 5 2.5 31 46

CP5-5-l(〇-Ac +/-) CP5-6-l(0-Ac -) (meg) (meg) _ 20 10 5 2.5 20 10 5 2.5 33 30 21 -12 -5 -12 -5 Data are reported as percent kill and by measuring the number of surviving CFUs in wells with bacteria, antibodies, complement and hl-60 cells at 6 min and in wells lacking antibodies but containing bacteria, complement and HL-60 cells The ratio of the number of surviving CFUs is calculated. 149159.doc -110- 201110977 Example 20: The immunogenicity of CP5 conjugates composed of high molecular weight polysaccharides in non-human primates (NHP) was observed to be stronger than low molecular weight polysaccharides for non-human primates (NHP) Studies were conducted to assess the immunogenicity of different laughing membrane conjugate formulations. Both formulations were tested at two different dose concentrations (2 and 2 〇 pg). The first formulation consists of a combination of high molecular weight (HMW) polysaccharide (about 130 kD) and CRMm. The second formulation contained a combination of low molecular weight (LMW) polysaccharides (about 25 kD) and CRM197. Five primates in each group were vaccinated with either single dose of vaccine and immune titers were monitored before vaccination and two weeks after vaccination. The OPA titer is defined as the serum dilution required to kill 40% of the S. aureus strain PFESA0266 in the 0PA assay. Antibody titers were also monitored by ELISA. It can be seen that the HMW vaccine is more active than the LMW formulation (Table 25). The antibody titer of the HMW vaccine is 3 to 10 times that of the lmv/vaccine. The OPA response rate of NHP receiving HMW vaccine was also higher (80% compared to 40%). Table 25: Immunogenicity of HMW polyacetate conjugate vaccine was observed to be stronger than LMW polysaccharide conjugate vaccine CP5-CRM197 dose (meg) / animal PD1* geometric mean blood OPA response rate HMW (125 kD) 20 32 80 —~ - 2 21 ' 80 - LMW(25 kD) 20 3 40 '~ 2 &quot;8 40 —~ * Increase the fold by comparing the CP5 ELISA titer with the vaccination & pre-validation rate 2 weeks after vaccination It was calculated based on the increase in OPA titer produced by monkeys 2 weeks after inoculation of a single dose of vaccine. Each group contained monkeys and the vaccine was formulated with AlP〇4 (250 μg/dose). ___ Further Summary Both of the binding chemistry described herein produced a serotype 5 capsular polysaccharide covalently linked to the carrier protein CRM197. The combination of the two methods produced 149I59.doc 201110977 free sugar, serotype 5 more than _ difference. The protein ratio and yield are not present at all levels of the art to which all of the disclosed inventions are mentioned in the specification. All of the methods used are incorporated herein by reference to the respective publications or patent applications. The disclosure of the present invention and the patent application are hereby incorporated by reference in their entirety as if individually and individually, in Details, but in the accompanying towel, you can still make some changes and improvements. [Simplified description of the diagram] Figure 1 shows the S. aureus serotype 8 intestine multi-repeated repetitive multidomain structure (N-acetamidoamine mannose (tetra) acid for ManNAeA, N acetyl ketone fucosamide for L -FucNAc, and N_acetinyl D_fucosylamine is d_FucNAc); Figure 2A shows the S. aureus serotype 8-in-a-tube (9) ethyl-branched analysis) and teichoic acid (phosphate analysis) Analysis of the fractions of ion exchange chromatography (Q_Sepharose); Figure 2B shows the dissolution of ion exchange chromatography (Q_Sepharose) of S. aureus serotype 8 capsular polysaccharide by two-way immunodiffusion analysis Figure 3 shows the effect of reducing the molecular weight of S. aureus serotype 8 capsular polysaccharide at 95 (under, during heat treatment) (Figures 3B, at pH 3.5, The temperature (55. 〇, 75. 〇 or 95C) during heat treatment reduces the molecular weight of S. aureus serotype 8 capsular polysaccharide 149159.doc • 112· 201110977 ring; Figure 4 shows the purified S. aureus serotype 8 The capsular polysaccharide is compared to the serotype 5 capsular polysaccharide during the heat treatment, respectively at the pH 3.5 and pH at pH 4.5 and varying with time at 95 ° C; Figure 5 shows mice receiving serotype 8 capsular polysaccharide-CRM197 conjugate compared to A1P04 treated control (circle) Survival rate increased; Figure 6 shows the repeating polysaccharide structure of S. aureus serotype 5 polysaccharide (#-acetamidoamine mannitol is ManNAcA, iV-acetyl L-fucosylamine is L-) FucNAc, and TV-acetyl-D-fucosylamine is D-FucNAcA); Figure 7A shows the S. aureus serotype 5 polysaccharide (Ο-acetamidine analysis) and teichoic acid (phosphate analysis) Analysis of the fractions of ion exchange chromatography (Q-Sepharose); Figure 7A shows the analysis of the dissociation of S. aureus serotype 5 multi-tone ion exchange chromatography (Q-Sepharose) by two-way immunodiffusion analysis; Figure 8A shows the effect of pH (3.5, 4 or 5) on reducing the molecular weight of S. aureus serotype 5 capsular polysaccharide at 95 °C during heat treatment; Figure 8B shows the temperature during heat treatment at pH 3.5 ( 55 ° C, 75 ° C or 95 ° C) to reduce the molecular weight of S. aureus serotype 5 capsular polysaccharide Effect; Figure 9 shows that pyelonephritis was reduced in mice receiving serotype 5 polysaccharide-CRM197 conjugate compared to the PBS-treated control group (shaded area was treated mice); Figure 10 shows high molecular weight inoculation (HMW) CP5-CRM, low molecular weight (LMW) CP5-CRM or PP5-CRM control in mice with golden yellow grapes 149159.doc • 113 - 201110977 cocci PFESA0266 after kidney recovery (CFU); Figure 11 shows a comparative mouse composition of OPA titers (geometric mean) of serum obtained from inoculated polysaccharide conjugates (high score CRM, low molecular weight (LMW) CP5-CRM). The mouse forming unit (HMW) CP5-ligand was obtained from 5 to 9 mice in each group. 149159.doc 114-

Claims (1)

201110977 VII. Patent application scope: 1. An immunogenic poly- vinegar-protein conjugate comprising a serotype 8 capsular polysaccharide of S. aureus awrews in combination with a carrier protein, wherein: a) the multi-enzyme The molecular weight is from 2 〇 kDa to 1000 kDa. 2. The immunogenic conjugate of claim 1, wherein the immunogenic conjugate has a molecular weight of from 200 kDa to 5 000 kDa. 3 _ An immunogenic polysaccharide-protein conjugate comprising an isolated S. aureus serotype 5 capsular polysaccharide in combination with a carrier protein, wherein: a) the polysaccharide has a molecular weight of from 2 〇 kDa to 1000 kDa. 4. The immunogenic conjugate of claim 3, wherein the immunogenic conjugate has a molecular weight of from 200 kDa to 5000 kDa. 5. The immunogenic conjugate according to any one of claims 1 to 4, wherein the polysaccharide has a molecular weight ranging from 70 kDa to 300 kDa. The immunogenic conjugate according to any one of claims 1 to 5, wherein the conjugate has a molecular weight ranging from 5 〇〇 kDa to 2500 kDa. The immunogenic conjugate according to any one of claims 1 to 4, wherein the polysaccharide has a ◦-acetylation degree of from 10〇/〇 to 100〇/〇. 8. The immunogenic conjugate of claim 7, wherein the polysaccharide has a degree of 0-acetylation of 50% to 1%/(( 9. The immunogenic conjugate of claim 7 wherein多糖 醯 醯 醯 醯 醯 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 149 In the phagocytic killing assay, the immunogenic compound is capable of producing a functional antibody according to the method of killing the bacterium. The immunogenic conjugate according to any one of claims 1 to 10, wherein the carrier protein is CRM197. The immunogenic conjugate of claim 11, wherein the CRMi97 is covalently linked to the polysaccharide via a urethane linkage, a guanamine linkage, or both. 13. As claimed in claim 11 or 12. An immunogenic conjugate wherein the molar ratio of the combination of lysine to CRM 97 is from about 1 :1 to about 25:1. 14. An immunogenic conjugate according to claim π, wherein at least the polysaccharide is present There is at least one covalent bond between cRMm# and more than every 5 to 10 brewing repeat units. 15. If the request item π or 14 is exempted a pro-conjugate, wherein at least one bond between the CRMm and the polysaccharide is present in every 5 sugar repeat units of the polysaccharide. 16. The immunogenic conjugate of any one of claims U to 15, wherein The CRMm comprises from 5 to 22 lysines covalently linked to the polysaccharide. The immunogenic conjugate of claim 16, wherein the CRMm comprises from 8 to 15 lysines covalently linked to the polysaccharide. 18. The immunogenic conjugate according to any one of items 1 to 17, wherein the immunogenic conjugate comprises less than 30% of a free 5 or 8 type polysaccharide, based on the total amount of the 5 or $ type polysaccharide. The immunogenic conjugate of claim 18, wherein the total immunoconjugate of the 5 or 8 type polysaccharide comprises less than 20% of the free 5 or 8 type polysaccharide. 149l59.doc 201110977 2 0 · An immunogenic composition comprising the immunogenic conjugate of any one of claims 1 to 9 and at least one adjuvant, diluent or carrier. 2 1 . The immunogenic composition of claim 20. Wherein the adjuvant is an aluminum-based adjuvant. 22. The immunogenic composition of claim 21, wherein the adjuvant The group consisting of aluminum phosphate, aluminum sulfate, and aluminum hydroxide. 23. The immunogenic composition of claim 21, wherein the adjuvant is aluminum phosphate. 24. The method of any one of claims 20 to 23 An immunogenic composition comprising less than 3% by weight of free 5 or 8 type, based on the total oxime of the 5 or 8 type polysaccharide. 25_Immunogenic composition of claim 24 , the total amount of 5 or 8 type vinegar is δ, and the oxime immunogenic composition contains less than 2 〇〇 /. Free 5 or 8 type polysaccharides. 26. A method of inducing an immune response against a S. aureus serotype 5 or serotype 8 capsular polysaccharide conjugate in an individual, the method comprising administering to the individual an immunologically effective amount as claimed in claim 2 An immunogenic composition according to any one of the preceding claims. 27. A method of producing an immunogenic polysaccharide-protein conjugate comprising a separate golden yellow grape sphere "clear type 8 capsular polysaccharide" in combination with a carrier protein, the method comprising the steps of: a) The isolated S. aureus serotype 8 capsular polysaccharide reacts with carbonyl di-triazine (CDT) in an organic solvent to produce an activated serotype 8 polysaccharide; and b) the activated serotype 8 polysaccharide and the carrier protein are organic In the solvent, 149159.doc 201110977 should produce a serotype 8 polysaccharide: carrier protein conjugate; wherein an immunogenic conjugate of the Saccharomyces cerevisiae serotype 8 infusion of the carrier protein binding is produced. - a method of producing an immunogenic polyacetic acid-protein conjugate comprising an isolated S. aureus gold: type 5 capsular polysaccharide in combination with a carrier protein, the method comprising the steps of: a serotype that activates a few bases 5 a) reacts the isolated S. aureus serum di-triterpene (CDT) in an organic solvent, a polysaccharide; and W 吏 the activated serotype 5 The sugar is reacted with the carrier protein in an organic solvent to produce a serotype 5 polysaccharide: carrier protein conjugate; wherein an immunogenic conjugate comprising a S. aureus ash clear type 5 capsular polysaccharide-carrier protein conjugate is produced. The method of claim 27 or claim 28, further comprising: lyophilizing the isolated polysaccharide; and resuspending the lyophilized polysaccharide in an organic solvent. 30. The method of any one of claims 27 to 29 The method wherein the activated isolated polysaccharide is first separated from the activation reaction and then reacted with the carrier protein. 3 1. The method of claim 3, wherein step b) comprises: 0 / Donggan the activation of the separation Separating the polysaccharide to produce a lyophilized activated polysaccharide; Η) drying the carrier protein to produce a lyophilized carrier protein; and U1) resuspending the lyophilized activated polysaccharide and the lyophilized carrier protein in an organic In the solvent, a mixture of the activated isolated polysaccharide and the carrier protein 149159.doc 201110977. The method of any one of claims 27 to 31, further comprising the step b) The mixture is diluted in a buffer and maintained at a pH of from about 8.8 to about 9.2 for at least 4 hours from about 2 Torr to about 26. 33. The method of any one of claims 27 to 31, further comprising the step of b) The reaction mixture is diluted in a buffer and maintained at a pH of about 9.0 at about 23 Torr for at least 4 hours. It further comprises isolating the 3 4. The method of any one of claims 27 to 33 is golden yellow. Staphylococcal serotype capsular polysaccharide-carrier protein conjugate wherein the organic solvent is a polar 3 5. The methodic aprotic solvent of any one of claims 27 to 34. 36. The method of claim 35, wherein the polar aprotic solvent is selected from the group consisting of disulfoxide (DMSO), dimercaptomethylamine (DMF), dimethylacetamide, iV-mercapto-2L The ketone and hexamethyl broken (tetra) amine (four) are called the group. 3 7 _ If § Qing seeking item 3 6 method, the pot φ 兮 兮 , , , , , , , 有机 有机 有机 有机 有机 有机 有机 有机 有机 有机 有机 有机 有机 有机 有机 有机 有机 有机 有机 有机38. If § Qing Qi 27 and 29 to 37 are in any way, the method of τ Shi Xiang, the method of reacting the polysaccharide with CDT includes determining the S. aureus serotype 8 in the laughing polysaccharide. The water content, and adjust the CDTiJl sound, * 5 w η is the end, until the organic solvent in the cDT: water molar ratio is about 1: i. 39. The method of any one of clauses 27 and 29, wherein the step of reacting the multi-brewed with the CDT comprises determining the water content of the S. aureus serotype 8 capsular polysaccharide and adjusting the CDT concentration to the organic solvent. Medium CDT Water 149159.doc 201110977 Mo ratio is about 0.5:1. 40. The method of any one of claims 27 and 29 to 38, wherein the step of reacting the polysaccharide with the CDT comprises determining the water content of the golden, glutinous serotonic capsular 8 polysaccharide. And adjusting the concentration of CDT, and the molar ratio of CDT:water in the main organic agent is about 0.75:1. The method of any one of claims 28 to 37, wherein the step of reacting the serotype 5 polysaccharide with the CDT comprises providing a CDT which is about 2 times more than the polysaccharide molar amount. . 42. In the case of claims 28 to 37 and 41, the method of 瑁 古 古 , 土 贝 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , . /〇 to 0.3〇/〇. 43. The method of claim 42, wherein the water concentration is adjusted to 〇2%. 44. The method according to any one of claims 29 to 43 wherein the step of isolating the activated serum-type polysaccharide comprises a dialysis transition. The method of any one of claims 27 to 44, wherein the carrier protein is diafiltered against NaCl before lyophilization, and the weight ratio of the NaCi/protein carrier protein is adjusted to about 〇. .5 to about 1 5. The method of any one of claims 27 to 45, wherein the carrier protein is CRM 1 97 0 47. The method of claim 46, wherein the activated serotype is reacted in a weight ratio of about 1:1 . The method of any one of clauses 27 to 47, wherein the isolated S. aureus serotype capsular polysaccharide is first mixed with imidazole or triazole and then mixed with CDT in an organic solvent. The method of any one of claims 27 to 48, further comprising hydrolyzing the serotype polysaccharide-carrier protein conjugate to remove unreacted active melons. 50. A method of making an immunogenic conjugate comprising an isolated S. aureus serotype 8 viburnum polysaccharide in combination with a wearer protein, the method comprising the steps of: a) allowing S. aureus serum Type 8 capsular polysaccharide and 3_(2_π-bipyridinium dithio)-propionate (PDPH) and carbodiimide in organic solvent=二应' to produce PDPH-linked polysaccharide; b) PDPH-linked polysaccharide and reduction The agent reacts to produce a polysaccharide of live sputum; c) isolates the activated serotype 8 polysaccharide to produce an isolated activated serum] 8 polysaccharide; β d) provides an activated carrier protein; e) activates the isolated activated serotype 8 polysaccharide The activated carrier protein reacts to produce a serotype 8 polysaccharide-carrier protein conjugate; thereby producing a S. serovar serotype method comprising the carrier protein and the carrier Staphylococcus serotype 5 capsule Separation of multi-mass combination 51. 8 immunoconjugate of capsular polysaccharide. A method for producing an immunogenic conjugate that binds to an isolated golden yellow sugar'. The method comprises the steps of: capsular polysaccharide and 3-(2-pyridyldiimide in an organic solvent, anti-a) to make golden yellow grapes Cocci serotype 5 dithio)-propanoid (PDPH) and carbonization should produce a PDPH-linked polysaccharide; 149159.doc 201110977 b) reacting the PDPH-linked polysaccharide with a reducing agent to produce an activated polysaccharide; c) isolating the Activating serotype 5 polysaccharide to produce an isolated activated serotype 5 polysaccharide; d) providing an activated carrier protein; e) reacting the isolated activated serotype 5 polysaccharide with the activated carrier protein to produce a serotype 5 polysaccharide-vector Protein conjugate; thereby producing an immunogenic conjugate comprising the isolated S. aureus serotype 5 capsular polysaccharide bound to the carrier protein. 52. The method of claim 50 or claim 51, wherein the activated carrier protein is isolated and the activated carrier protein is reacted with the activated polysaccharide. The method of any one of the items 50 to 5, wherein the step c) further comprises lyophilizing the isolated activated serotype 8 polysaccharide to produce a lyophilized activated serotype polysaccharide. The method of any one of clauses 50 to 53, wherein the organic solvent is a polar aprotic solvent. The method of claim 54, wherein the polar aprotic solvent is selected from the group consisting of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide, hydrazine methyl-2-pyrrolidine a group consisting of a ketone and a hexamethylenephosphonamide (HMpA). The method of claim 55, wherein the polar aprotic solvent is dimethyl sulfoxide (DMSO), such as the method of any one of claims 5G to 56, wherein the carbodiimide is 149159.doc 201110977 1- Ethyl-3-(3-didecylaminopropyl)-carbodiimide (EdaC). The method of any one of claims 50 to 57, wherein the step of reacting the serotype lipopolysaccharide with PDPH and EDAC in an organic solvent comprises maintaining a polysaccharide of about 1:5:3: PDPH.EDAC weight ratio . The method of any one of claims 50 to 58, wherein the reducing agent is dithiothreitol (DTT). The method of any one of claims 50 to 59 wherein the activation of the carrier protein comprises reacting the carrier protein with bromoacetic acid. 61. The method of claim 60, wherein the bromoacetic acid comprises hydroxybutylide bromoacetate (BAANS). The method of any one of claims 50 to 61, wherein the step of isolating the activated serotype polysaccharide comprises diafiltration. The method of any one of claims 50 to 62, wherein the step of isolating the activated carrier protein comprises diafiltration. The method of any one of claims 5 to 63, further comprising the step of hydrolyzing the serum-type polysaccharide-carrier protein conjugate to remove unreacted reactive groups. 65. The method of Moon Length 64, wherein the step of hydrolyzing the serotype of the multi-flavored carrier protein conjugate comprises adding cysteamine hydrochloride. The method of any one of claims 50 to 65, further comprising isolating the i ', axon". The mouthpiece comprising the isolated S. aureus serotype capsule in combination with a carrier protein The method of claim 66, wherein the serotype polysaccharide: carrier protein conjugate &amp; isolation method comprises diafiltration. 149159.doc 201110977. The method of any one of claims 50 to 67, wherein the carrier The protein is CRM 1 97 〇69. The method of claim 68, wherein the activated serotype polysaccharide is reacted with (: 尺)^97 in a weight ratio of about 1:1. 70. according to any one of claims 27 to 69. The method of claim 7, wherein the activated polysaccharide has a molecular size of from about 20 kDa to about 1 〇〇〇 kDa. 71. The method of claim 70, wherein the activated polysaccharide has a molecular size of from about 5 〇 kDa to 500 kDa. The method of any one of claims 27 to 71, wherein the immunogenic polysaccharide-protein conjugate has a molecular size of from 4〇〇kDa to about 5000 kDa. 73. An immunogenic conjugate, such as The method of any one of claims 27 to 72 is produced. 74. An immunogenic conjugate comprising more than about 1% and less than about 30% free polysaccharide. 75. The immunogenic conjugate of claim 73, comprising less than about 2% free vinegar. An immunogenic composition comprising the immunogenic conjugate of any one of claims 73 to 75 and at least one adjuvant, diluent or carrier. 77. The immunogenic composition of claim 76 The adjuvant is an adjuvant based on the above. 78. The immunogenic composition of claim 77, wherein the adjuvant is selected from the group consisting of aluminum sulphate, aluminum sulfate, and aluminum hydroxide. An immunogenic composition of 78, wherein the adjuvant is a dish of acid. In an immunogenic composition according to any one of claims 76 to 79, each type is 149159.doc -10- 201110977 sugar In total amount, the immunogenic composition comprises less than 30% of the free polysaccharides. 8 1 . The immunogenic composition of claim 80, based on the total amount of each type of polysaccharide, the immunogenic composition comprising Less than 20% of free polysaccharides of various types. 82. A golden yellow grape covalently bound to carrier protein The capsular polysaccharide of the bacterium 8 has a molecular weight of 2 〇 kDa to 1000 kDa; wherein the combined molecular weight of the covalent conjugate with the carrier protein is about 200 kDa to 5000 kDa. The capsular polysaccharide having a molecular weight of 2〇kDa to 1〇〇〇kDa covalently bound to the carrier protein, wherein the molecular weight of the covalent conjugate of the multi-flavored carrier protein is It is about 200 kDa to 5000 kDa. 84. The covalent attachment of claim 82 or claim 83 to the carrier protein wherein the polysaccharide has a molecular weight ranging from 7 〇 kDa to 300 kDa. 85. The covalent bond of claim 80 or claim 83 to the carrier protein has a molecular weight ranging from 500 kDa to 2500 kDa. 86. A polysaccharide covalently bound to a carrier protein of claim 82 or claim 83, wherein the carrier protein is CRM197. .87. The polysaccharide covalently bound to the carrier protein of claim 86, wherein the CRM 97 is covalently attached to the polysaccharide via a urethane linkage, a guanamine linkage, or both. 88. As claimed in claim 86 or claim "covalently bound to the carrier protein, the molar ratio of the combined amidoxime to CRM197 is from about 1 :1 to about 25:1 〇149159.doc 201110977 89_ A polysaccharide covalently bound to a carrier protein as claimed in claim 88, wherein at least one covalent bond between CRM197 and the polysaccharide is present in at least every 5 to 10 sugar repeat units of the polysaccharide. 88 or the covalent attachment of claim 89 to the listener of the carrier protein wherein there is at least one bond between CRM197 and the polysaccharide in every 5 sugar repeat units of the polysaccharide. 91. In claims 88 to 89 Any one of the covalently bound to the carrier protein, wherein the CRM 9-9 comprises 5 to 22 lysines covalently linked to the polysaccharide. 92. Covalently bound to the carrier protein as claimed in claim 91 a polysaccharide, wherein the CRM 97 comprises from 8 to 15 lysines covalently linked to the polysaccharide. 93. An immunogenic composition comprising a total mussel combination as claimed in claim 82 to % To the polysaccharide of the carrier protein and at least one adjuvant, diluent or carrier. The immunogenic composition of claim 93, wherein the adjuvant is an aluminum-based adjuvant. 95. The immunogenic composition of claim 94, wherein the adjuvant is selected from the group consisting of phosphoric acid, aluminum sulfate, and The immunogenic composition of claim 95. The immunogenic composition of claim 95, wherein the adjuvant is aluminum phosphate. 97. a species that reduces or prevents staphylococcal infection in a subject, a disease or condition associated with staphylococci The method comprising the step of administering to the individual an immunologically effective amount of the immunogenic composition of any one of claims 20 to 25 and 76 to 81 and 93 to 96. 98. The method of claim 97 Wherein the infection, disease or condition is selected from the group consisting of 149159.doc •12·201110977 j Staphylococcus aureus, sepsis and a genus. The species is reduced or prevented from staphylococcal infection in an individual undergoing a surgical procedure. The method of the present invention comprises the step of administering to the individual an immunologically effective amount of the immunogenic composition of any one of claims 20 to 25 and 76 to 81 and 93 to 96 prior to the surgical procedure. The method of any one of items 97 to 99, The individual is a human, domestic pet or livestock. ιοί. A method for isolating a S. aureus capsular polysaccharide having a molecular weight of 20 to 1 〇〇〇 kDa, the method comprising the step of subjecting the bacterial cell suspension to an acidic heat treatment. 102. An antibody produced by the capsular polysaccharide, immunogenic conjugate or immunogenic composition of the invention. 103' The antibody of claim 1 〇 2 wherein the antibody is efficacious or phagocytic The antibody was functional in the sex kill assay as measured by killing bacteria. 149159.doc